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who has a patent?!?!?!

Just like you want to know how many NIH grant the guy down the hall has, you’re probably also wondering if he has any patents.  While you toil away at the bench or in the clinic, this dude is probably raking in the dough from his patent on the little floaty things that hold eppendorf tubes in the hot water bath.  Do you want to find out?  Then go straight to the US patent office website and do a search!  This will take you to the advanced search page and just type in: IN/[LastName]-[Firstname] (for example: IN/doe-john) or you can leave the first name off (for example: IN/doe).  You might be shocked at what you find…

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flow cytometry protocols, facilities and online protocols

Here’s a cool compilation of online flow cytometry resources offered by flow facilities from around the world that I found on website of the flow cytometry core at the Salk Institute in San Diego. For future reference, I’ve added this table to the flow cytometry page under Lab Resources and will continue to update it as I get more information. If your university has a good flow cytometry core website, please let me know and I’ll add it to the list. From my own experience, I would recommend trying the Purdue University links first…

Home Page Institution More Links Location
MyCyte.org MyCyte.org [Forum]

[Blogs] [RSS Feeds] [News] [Events] [Links/Sites] [Products] [Standards] [Education] [Cytometry Abstracts]

US-OR
Purdue Purdue University Cytometry Laboratories [Cytometry Mailing List]

[Flow Cytometry Software]

[CD-ROMs project]

[Links/Sites]

[Protocols]

[Suppliers]

[Confocal/Microscopy links]

[Meetings]

[Courses]

[Rates Summary Table]

[Lecture Slides]

[Education/Books]

[Images]

[Jobs]

US-IN
Cancer Research UK LRC Lincoln's Inn Fields [Cell cycle analysis]

[Apoptosis]

[Functional studies]

[Sorting]

[Multicolour]

[Stem cell analysis]

[Data analysis]

[Links/Sites]

[Laser Scanning Cytometry]

[Flow Club]

UK
JCSMR John Curtin School of Medical Research, Canberra [Tutorials][Data Analysis][Statistics][Software:Facsimile][Facscan Tutorial test][LSR][Literature][Protocols][Links/Sites][Rates] AU
Albert Einstein Albert Einstein College of Medicine Flow Core [About flow][Instrumentation][Fluorophores][Fluorescent Proteins][Protocols][Links/Sites][Rates] US-NY
CytoRelay MPI-Biochemie, Martinsried, Germany:Cell Biochemistry [Links/Sites][Suppliers][Cell Function][Aquatic Links][Software links/Mailing Lists/FAQs][Education/Books][Consensus/Ring Trials][Microorganisms][Purdue mailing list mirror] DE
WEHI WEHI Cytometry Lab [Overview] [Links/Sites][Fluorescence][Fluors][Sorter Comparison][Rates][FCMDesigner (Java)][Software:WEASEL][Links/Sites][Publications] AU
Aberystwyth University of Wales Institute of Biological Sciences [Links/Sites][Publications][Instrumentation][Software][Sample Prep][Microcyte cytometer] [Microbiology] UK
Cytonet UK Cardiff, UWCM [Flow basics][RMS Cytometry][UK Jobs/Notices][UK Meetings][Software:Cylchred][Links/Sites][Suppliers] UK
Salk CCMI Salk Institute for Biological Studies CCMI [Links/Sites][Compensation Howto][Fluorochrome Table][Protocols][Lasers][Software] US-CA
TSRI Scripps Research Institute Flow Core [Home][Turbosort][Sort Recovery Calculation][FACStar] / [Vantage jet optimization][Protocols][Software:WinMDI][Sites/Links] US-CA
IACF Immunology Applications Core Facility, University of Chicago [Technical protocols][Sort prep][Protocols][References][Rates] {Flash required} US-IL
Prague Centrum Pr?tokové Cytometrie [Basics][Sorting][Data analysis][Instrumentation][Cytometers][Rates][Protocols (Czech)][Links/sites] CZ
St Mary's Imperial College, London [PowerPoint presentations][Rates][Sorting][Flow Course] UK
NCI ETI NCI ETI Branch Flow Core [Protocols][Spectra][Sorting][Facscan/Calibur info][Advanced projects/protocols][Links/Sites] US-MD
UTMD MD Anderson Cancer Center Cell and Tissue Analysis Core [Flow][Protocols][Quantitative][Side Population][More Protocols][Compensation][Links/Sites] US-TX
Berkeley UC Cancer Research Lab [Flow Basics][Compensation][Rates][Instrumentation][links/Sites] US-CA
HSS Fannie E. Rippel Foundation [Instrumentation][Rates] US-NY
ISAC International Society for Analytical Cytology [Cytometry journal][Conferences][Tutorial material][FCS 3.0][Biosafety][Software] US
Fluorophores.org Fluorescent dyes database [About][Browse][Links/Sites] Austria
Olomouc Institute of Experimental Botany Molecular Cytogenetics and Cytometry [Plant DNA FCM][Flow Cytogenetics][Plant protocols][Reagents][Suppliers] CZ
Roswell Park Roswell Park Cancer Institute Laboratory of Flow Cytometry [About][Software][Services/Rates][Instrumentation][Courses/Powerpoint slides][Programs/Outreach][Links/Sites] US-NY
Herzenberg Lab at Stanford Herzenberg Laboratory [Protocols][FACS Development Group] US-CA
Stanford Stanford Shared FACS Facility [Flow overview][Rates][SOPs]][Instruments/Fluors][Documentation][User Guides] US-CA
UIowa Holden Comprehensive Cancer Center at UIowa [Flow fundamentals][Protocols][Rates] US-IA
Vanderbilt Vanderbilt HHMI Flow Cytometry Facility [Overview] [Rates] [Forms/Protocols][Links/Sites][Overview] US-TN
Baylor Baylor Flow Cytometry Core [Instruments][Rates][Protocols][Links/Sites] US-TX
Beth Israel Flow Cytometry and Sorting Facility [Core][Policies][Rates][Links/Sites] US-MA
Bremen Max Planck Institute for Marine Microbiology [Overview][Links/Sites] DE
CTEGD University of Georgia [Overview] [Instruments] [Rates] [Links/Sites] US-GA
HUGTiP Hospital Universitari Germans Trias i Pujol [Applications][Links/Sites][Rates][Journals/Books] ES
IMMAG Medical College of Georgia Institute of Molecular Medicine and Genetics [Instrumentation][Guidelines][Rates][Links/Sites] US-GA
Kiel Forschungs- und Technologiezentrum Westküste [Phytoplankton][Flow Overview][Sorting Overview][Links/Sites] DE
LSU Louisiana State University School of Veterinary Medicine [Instruments][Rates][Protocols][Links/Sites] US-LA
Penn SDM University of Pennsylvania School of Dental Medicine [Flow Primer][Protocols] [Links] [Rates] US-PA
AEFCG Australian Environmental Flow Cytometry Group [Research] AU
SUNY USB Research Flow Cytometry Core [Overview] [Rates][Links/Sites][Clinical] US-NY
UAMS University of Arkansas Microbiology and Immunology Flow Core [Overview][Cytometric Bead Array][Biohazard Review Form][Sample Submission Form] US-AR
UCLA Janis V. Giorgi Flow Cytometry Core [Protocols][Biosafety][Rates][Policies] US-CA
UPCI University of Pittsburgh Cancer Institute [Worksheets][Rates] US-PA
Cornell Cornell Biomedical Sciences Flow Cytometry Core [Protocols][Sample Prep][Rates][Software] US-NY
Bern University of Bern Department of Clinical Research Flow Cytometry Lab [Overview][Links/Sites][Flow Course] CH
Bigelow J.J. MacIsaac Facility for Aquatic Cytometry [General][Aquatic][Plankton] US-ME
Calgary University of Calgary Faculty of Medicine [Overview][Links/Sites][Rates] CA
Cooper Hospital/UMC Camden, New Jersey [Methods][Reference Ranges][Publications] US-NJ
Cornell Cornell University Flow Cytometry Facility [Overview] [Rates][Links/Sites] US-NY
Hopkins Immunology Johns Hopkins Immunology [Analyzer guidelines][Sorting guidelines][Contact][Billing][Links/sites] US-MD
Hopkins CFAR Johns Hopkins Center for AIDS Research [Overview][FAQ] [Links/Sites] US-MD
Iowa State Cell and Hybridoma Facility [Protocols][Sites/Links] US-IA
Mario Roederer Mario Roederer's Home Page [Compensation Tutorial][Antibody Conjugation] US-CA
McMaster McMaster University Flow Facility [Links][Facscan Operation][Rates] CA
MIT Flow Cytometry Core Facility [Rates][Protocols][Links] US-MA
Newcastle University of Newcastle Department of Surgery [Description] [Research] [Courses] [Links] UK
NIAMS IRP National Institute of Arthritis and Musculoskeletal and Skin Diseases [Protocols] [FRET] [Links/Sites] US-MD
OHRI Ottowa Health Research Institute [Overview] [Rates] [Links/Sites] CA
Prague Institute of Botany, AS CR [Plant FCM][Plant DNA][Protocols][Links/Sites] CZ
Princeton Princeton University Flow Cytometry Core [Instruments][Protocols][Links/Sites] US-NJ
Rockerfeller FCRC Flow Cytometry Resource Center [Rates][Operation/Tips][Instruments] US-NY
Roscoff Station Biologique de Roscoff, France [Phytoplankton][Software:CytoWin] FR
RRC University of Illinois at Chicago [Description] [Protocol links] [Rates] US-IL
Sheffield University of Sheffield Division of Genomic Medicine Core Cytometry [Rates] [Protocols] [Links/Sites] UK
TIG Therapeutic Immunology Group, Dunn School, Oxford [Protocols] [Tutorials] [Instrument operation] UK
UCHC UCONN Health Center Flow Facility [Flow description][Links/Sites][Rates] US-CT
UMN University of Minnesota Cancer Center [Protocols][links/Sites][Instrument operation] US-MN
Urbana-Champaign University of Illinois Biotechnology Center Flow Cytometry Facility [Cytokines] [Apoptosis] [Links] US-IL
UVa University of Virginia Flow Cytometry Core Facility [Protocols][FAQs][Rates] US-VA
Amsterdam Microscopy and Cytometry Software: Ron Hoebe [Software:Flow Explorer][Refresh Ratio][List Math] NL
NECyt New England Cytometry [Tutorials/Presentations][Biosafety][Links/Sites][Jobs][BBG][Mailing List][Meetings] US-MA
Caltech Caltech Flow Cytometry Facility [Policies/Rates][Links/Sites] US-CA
EOSHI/CINJ Rutgers Environmental and Occupational Health Sciences Institute [Description][Rates] US-NJ
HMDS Leeds Leeds Flow Cytometry [Flow Overview][Fluorochromes] UK
Frank J. Jochem Dr. Frank J. Jochem [Oceanography][Phytoplankton] DE
Keith Bahjat Antibody Cross Reactivity Resource [Antibody Crossreactivity Resource] US-FL
Newcastle FACS University of Newcastle Biomolecular Research Facility [Description] [Rates] AU
NFCR National Flow Cytometry Resource [Overview/Research] US-NM
Novosibirsk Institute of Chemical Kinetics and Combustion, Novosibirsk, Russia [Scanning Flow Cytometry][Flying Light Scattering] RU
UCSD UCSD Cancer Center Flow Cytometry Shared Resource [Protocols][Rates] US-CA
Umass/Amherst Facility closed 2000 [About][MFI Software] US-MA
UMass/Worcester Core Flow Lab [Instrumentation][Rates][Links/Sites][Protocols] US-MA
UNM Cytometry Bioengineering Consortium [Bioengineering Consortium][High Throughput] US-NM
UNM University of New Mexico Shared Flow Cytometry Resource [High Throughput][Rates] US-NM
Utah cores.utah.edu [Sorting][Rates][Links/Sites] US-UT
UWashington Immunology University of Washington Immunology Cell Analysis Facility [Protocols][Rates] US-WA
Birmingham University of Birmingham Animal Cell Technology Group [Links/Sites] UK

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non-r01 nih grants for new investigators

So if you’re starting out as a new investigator, you’re probably not going to get an R01 grant right off the bat.  There are, however, many NIH grants that are specifically aimed at new investigators for career development as a stepping stone to future application for an R01. 

I have a few buddies who are going through this process now and applying for many of these.  For those of you who aren’t up to this point yet, it may be useful to get familiar with some of these grants–at least know what they are referring to.  You will hear these terms being thrown around a lot in conversation and more importantly, it won’t be too long before this will be useful information for you to know. 

Code Description
R03 NIH Small Grant Program

  • Provides limited funding for a short period of time to support a variety of types of projects, including: pilot or feasibility studies, collection of preliminary data, secondary analysis of existing data, small, self-contained research projects, development of new research technology, etc.
  • Limited to two years of funding
  • Direct costs generally up to $50,000 per year
  • Not renewable
  • Utilized by more than half of the NIH ICs
  • See parent FOA
R15 NIH Academic Research Enhancement Award (AREA)

  • Support small research projects in the biomedical and behavioral sciences conducted by students and faculty in health professional schools and other academic components that have not been major recipients of NIH research grant funds
  • Eligibility
  • Direct cost limited to $150,000 over entire project period
  • Project period limited to up to 3 years
  • All NIH ICs utilize except FIC an NCMHD
  • See parent FOA
R21 NIH Exploratory/Developmental Research Grant Award

  • Encourages new, exploratory and developmental research projects by providing support for the early stages of project development. Sometimes used for pilot and feasibility studies.
  • Limited to up to two years of funding
  • Combined budget for direct costs for the two year project period usually may not exceed $275,000.
  • No preliminary data is generally required
  • Most ICs utilize
  • See parent FOA
K01
Mentored Research Scientist Development Award (K01)
  • This omnibus NIH K01 program is supported by NHGRI, NIA, NIAAA, NIAID, NIAMS, NIBIB, NICHD, NIDCD, NIDDK, NIDA, NIEHS, NIMH, NINDS, NINR, NCCAM, NCRR, and ODS. The purpose of the K01 program is to provide support and “protected time” (3-5 years) for an intensive, supervised career development experience in the biomedical, behavioral, or clinical sciences leading to research independence. Awards are not renewable, nor are they transferable from one principal investigator to another.

The Bernard Osher Foundation/NCCAM CAM Practitioner Research Career Development Award (K01)

  • This program is supported by NCCAM. The purpose of this K01 is to provide research training support for CAM Practitioners with clinical doctorates, who have had limited opportunities for research training, but a strong desire to pursue a career in CAM research.

NCI Mentored Research Scientist Development Award to Promote Diversity (K01)

  • The NCI invites K01 applications from individuals representative of groups that have been shown to be underrepresented in health-related science, who have been recipients of an NIH Research Supplement to Promote Diversity Award, any NRSA (individual F31/F32 or institutional T32), or can demonstrate that they have been supported in a mentored capacity within any research grant equivalent to an NIH peer-reviewed research grant.

NIDDK Mentored Research Scientist Development Award (K01)

  • The NIDDK invites K01 applications from advanced postdoctoral and/or newly independent research scientists (usually with a Ph.D. degree) in biomedical or behavioral sciences who are pursuing careers in research areas supported by the NIDDK.

NINDS Career Development Award to Promote Diversity in Neuroscience Research (K01)

  • Supported by NINDS, the objective of this program is to promote diversity among faculty-level neuroscience investigators who are competitively funded to conduct independent research.

NINR Mentored Research Scientist Development Award for Underrepresented or Disadvantaged Investigators (K01)

  • The purpose of this NINR K01 is to encourage the development of qualified underrepresented or disadvantaged nurse scientists to become independent investigators in research settings.
K02
Independent Scientist Award (K02)
  • This omnibus NIH K02 program is supported by NHLBI, NIA, NIAAA, NIAID, NICHD, NIDCD, NIDCR, NIDA, NIEHS, NIMH, NINDS, and ODS. The K02 provides support for newly independent scientists who can demonstrate the need for a period of intensive research focus as a means of enhancing their research careers. The K02 is intended to foster the development of outstanding scientists and to enable them to expand their potential to make significant contributions to their field of research.

K08
Mentored Clinical Scientist Research Career Development Award (K08)
  • This omnibus NIH K08 program is supported by NCI, NEI, NHLBI, NIA, NIAAA, NIAID, NIAMS, NIBIB, NICHD, NIDCD, NIDCR, NIDDK, NIDA, NIEHS, NIGMS, NIMH, NINDS, NCCAM, and ODS. The K08 represents the continuation of a long-standing NIH program that provides support and “protected time” to individuals with a clinical doctoral degree for an intensive, supervised research career development experience in the fields of biomedical and behavioral research, including translational research. Individuals with a clinical doctoral degree interested in pursuing a career in patient-oriented research should refer to the NIH Mentored Patient-Oriented Research Career Development Award (K23).

NCI Mentored Clinical Scientist Research Career Development Award to Promote Diversity (K08)

  • This NCI-sponsored K08 award is specifically designed to promote career development of racially and ethnically diverse individuals who are underrepresented in health-related science and for those who are committed to a career in cancer health disparities, biomedical, behavioral or translational cancer research.
K22
K23
Mentored Patient-Oriented Research Career Development Award (K23)
  • This omnibus NIH K23 program is supported by NCI, NEI, NHLBI, NIA, NIAAA, NIAID, NIAMS, NIBIB, NICHD, NIDCD, NIDCR, NIDDK, NIDA, NIEHS, NIGMS, NIMH, NINDS, NINR, NCCAM, and ODS. The purpose of the K23 is to support the career development of investigators who have made a commitment to focus their research endeavors on patient-oriented research. Clinically trained professionals or individuals with a clinical degree who are interested in further career development in biomedical research that is not patient-oriented should refer to the Mentored Clinical Scientist Career Development Award (K08).

NCI Mentored Patient-Oriented Research Career Development Award to Promote Diversity (K23)

  • The NCI announce the availability of the K23 award for career development of ethnically and racially diverse individuals with a health professional doctoral degree from groups that have been shown to be underrepresented in health-related science.

If any of the readers have had experience with these grants or applying for them, please share your experience or any tips that you may have to offer in the comments…

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CRISP – the nih grant database

Be honest, you want to know what NIH grants the PI down the hall has.  Right?  Of course you do.  You want to know how good he’s got it and how that compares to your NIH funding situation.  Well, the completely open thing to do would be to ask but then you look nosey and the other PI might figure out your ulterior motive.  But you have another option: the CRISP database.

To quote from the CRISP website:

CRISP (Computer Retrieval of Information on Scientific Projects) is a searchable database of federally funded biomedical research projects conducted at universities, hospitals, and other research institutions. The database, maintained by the Office of Extramural Research at the National Institutes of Health, includes projects funded by the National Institutes of Health (NIH), Substance Abuse and Mental Health Services (SAMHSA), Health Resources and Services Administration (HRSA), Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDCP), Agency for Health Care Research and Quality (AHRQ), and Office of Assistant Secretary of Health (OASH). Users, including the public, can use the CRISP interface to search for scientific concepts, emerging trends and techniques, or identify specific projects and/or investigators.

So you can use this database to spy on any investigator’s NIH funding status.  But in all seriousness, the CRISP database can be a really useful tool if you are thinking of writing a grant or even starting a new project because you can see if someone is already funded to work on your project.  It can potentially save you a lot of time in not writing a grant that someone else already has (or at least give you an opportunity to sufficiently distinguish your own grant).  Moreover, by knowing who else is working on a similar project, you can either know who to contact for collaboration or help (if you need it) or in contrast, you’ll know who to hide your work from…

Addendum:

Thanks to the readers who pointed out that CRISP is no longer up and running.  But, for those of you who still have the morbid curiosity to see which cocksuckers have NIH funding while you languish away barely making ends-meet on foundation grants, there is the RePORT Expenditures and Results query tool at: http://projectreporter.nih.gov/reporter.cfm

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nih grant codes – is k08 a grant or a type of submarine ?

How many times have you heard people talking about R01 grants or K22 grants and you’re like, “what the hell is he talking about?”  A lot for me.  Many years went by in medical and graduate school where I would hear people talking about these different NIH grants without any clue about what each grant was for.  Eventually I learned about some of them through just talking to people but then I recently discovered the internet and looked up this comprehensive table at the NIH website.  If you want to sound intelligent or be able to participate in conversations about the K02 independent scientist award, check out this site:

http://grants.nih.gov/grants/funding/ac_search_results.htm

Sooner or later if you stay in academia, you’re gonna have to get awfully familiar with these grants…

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advances in cancer genomics webinar

From the Science/AAAS:

Watch Live on Thursday, April 30, 2009
12 noon Eastern, 9 a.m. Pacific, 4 p.m. GMT

Cancer is a complex family of diseases, characterized by the deregulation or dysregulation of the normal control pathways for cellular growth and/or apoptosis. Traditional research programs have focused on identifying and quantifying environmental and inherited factors associated with cancers found in particular tissues. Despite many advances, these approaches have historically been limited in scope due to technological limitations or excessive cost. With next generation genomic platforms, scientists are now able to cost-effectively assay individual cancer genomes and characterize them in terms of the global genetic, epigenetic, and transcriptional changes. In depth characterization of these events—and the relationships between them—will lead to better understanding of the mechanisms of tumorigenesis, metastasis, and therapeutic response. In this timely webinar, a panel of distinguished scientists will share their latest advances in cancer genomics and offer their views on the road ahead for this important area of research.

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invitrogen promotion

In case anyone could use it, Invitrogen is offering a promo until March 20th, 2009 where they will give you a $20 VISA gift card if you buy $450 worth of supplies from them online.  Our lab used to regularly drop tons of money with Invitrogen, so I figure that someone out there could use this. 

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PCR resources

So one of the bread and butter techniques in science is PCR.  And while it can be oh so easy, there are a surprising number of subtleties to each of those steps we take for granted.  That is, until our PCR doesn’t work and we’re left wondering why.  The worst is when your PCR has worked for 3 months and then all of a sudden it doesn’t work anymore and you’re sitting there saying, WTF?!?!?!?  Moreover, PCR has gotten increasingly sophisticated with many different variants compared to what it was like when I started.  (When I started, RT-PCR was the cutting edge–yes, I’m that old).

Anyway, in an effort to help out with protocols and troubleshooting, I have assembled a number of online resources which I have found to be useful. 

PCR Links.com contains general information (on primers, polymerases, inhibitors, additives, optimization and troubleshooting), online books, as well as information (including protocols) on various types of PCR (including AFLP, Alu-PCR, Asymmetric PCR, Colony PCR, DD-PCR, Degenerate PCR, Hot-start PCR, in situ PCR, inverse PCR, Long-PCR, Multiplex PCR, Nested PCR, PCR-ELISA, PCR-RFLP, PCR-SSCP, QC-PCR, RACE, RAPD, real-time PCR, Rep-PCR, RT-PCR, TAIL-PCR, Touchdown PCR and Vectorette PCR).

From the Real-time quantitative PCR resource at the Iowa State University, attached are an-introduction-to-real-time-pcr-qpcr-assay-design-and-optimization and example-of-an-entire-qpcr-setup.

One good general website for PCR protocols is Protocol-Online for PCR (including protocols for everything from standard PCR, RT-PCR and quantitative real-time PCR to more specialized variants of PCR).

From Yale University, check out Octavian Henegariu’s website for PCR for valuable information on protcols and troubleshooting.  I have attached pdf printouts from this website on general PCR guide, choosing/designing PCR primers, designing PCR programs, magnesium concentration, dNTP concentrations, taq polymerases and PCR troubleshooting.

From the University of Helsinki, FastPCR–a free software package that has many capabilities including (from the FastPCR website): 

•    Standard, inverse, long, real-time PCR analysis – identification of the optimal primers for PCR, hybridization, or sequencing;
•    Multiplex PCR primers design – fast primer design with a cross-dimer test for high sensitive multiplex PCRs;
•    Group-specific PCR primers design – design of universal PCR primers for all sequences (there is no need for a multiple DNA alignment);
•    Unique PCR primers design – design of specific PCR primers for each sequence;
•    Degenerate PCR – primers design directly on an amino acid sequence;
•    Automatically SSR loci detection and direct PCR primers design;
•    In silico PCR and probe search – prediction of probable PCR products and search of potential mismatching location of the specified primer(s)/probe (s);
•    TaqMan and Molecular Beacons probes and other probes design;
•    LUX (self-quenched) primers design for quantitative PCR;
•    Self-Reporting DNA/DNA primers for qPCR analysis;
•    Primer Secondary structure analysis – self-dimer and cross-dimer primer analyses; primer alignment and melting temperature calculation;
•    False priming analysis -  (the secondary (non-specific) binding test) primers checking for multiple annealing sites using sequence alignment algorithms;
•    Primer quality report - a unique way for PCR efficiency determination;
•    Comprehensive primer report – comprehensive pairs and individual primers analysis (Tm and dimer detection);
•    Multitask PCR primers and probes design - simultaneously design primers or probe with different parameters and for different targets within the same sequence; interaction different tasks.
•    Repeats search: Invert, Direct, Simple and others;
•    MITE elements search;
•    LTR-retrotransposons search – discovery new LTRs, clustering and whole elements discovery;
•    SSR (Simple Sequence Repeat) locus search – two, three, four or five perfect and imperfect core motif
•    Clustering sequences (BLAST2 related alignment);
•    Sequence alignment – using universal degenerated code;
•    Complement, reverse and reverse-complement strand modification;
•    Consensus sequence for two or mores sequences;
•    Extraction of specific fragments from a larger sequence;
•    DNA-to-Protein translation and Protein-to-DNA reverse translation;
•    Calculation of the annealing temperature of PCR (for unknown PCR products);
•    Database analysis tools;
•    Restriction analysis;
•    Tools: patterns analysis – CG% content; purine-pyrimidine ration; GC skew (G-C)/(G+C); the melting temperature and linguistic sequence complexity;
•    Sequence complexity profile calculation -  fast calculation of linguistic sequence complexity of DNA or protein sequences with sliding windows from 10 to 2000 characters (this approach provided an efficient way to detect  – SSR loci, introns-exons, S/MARs (Scaffold/Matrix Attachment Regions) or else);
•    Pair Sequences Similarity – calculation the similarity for entered sequences in alignment format like MEGA or GCG/MSF and also non alignment raw formatted sequences.

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scansite: an online resource for detecting motifs of protein kinase phosphorylation

Scansite was developed by Michael Yaffe, a world reknowned cell signaling (in particular, protein phosphorylation) researcher at MIT.  Scansite is an online resource to search for motifs within proteins that are likely to be phosphorylated by specific protein kinases or bind to domains such as SH2 domains, 14-3-3 domains or PDZ domains.

Optimal phosphorylation sites for particular protein Ser/Thr kinases or protein-Tyr kinases are predicted using the matrix of selectivity values for amino acids at each position relative to the phosphorylation site as determined from the oriented peptide library technique described by Songyang et al., 1994, Current Biology 4, 973-982 and Songyang et al., 1995, Nature 373, 536-539.

Optimal binding sites for SH2 domains, PDZ domains, 14-3-3 domains and other domains are determined using the matrix of selectivity values for amino acids at each position relative to an orienting residue as determined by the oriented peptide library technique described in Songyang et al., 1993 Cell 72, 767-778, Songyang et al., 1997 Science 275, 73-77 and Yaffe et al., 1997 Cell 91, 961-971.

The same matrices of selectivity values are used in an approach to provide relative scores of candidate motifs in protein sequences evaluated.  Proteins can be scanned by accession number or with an input sequence.   One or many proteins can be scanned at a time.  Scansite will also allow you to scan the genomes of various species for sequences that are compatible with over 60 protein kinase recognition sites. 

If you are interested in or work on protein phosphorylation, check out Scansite!

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to my regular readers

Sorry–it’s been a slow week for new content on mudphudder’s blog.  This has been a really busy week and I’m halfway home now.  I’ll write put some new material up this weekend and ask that you bare with me for the next two days.  It should be smooth sailing from there on out.

For now, if you haven’t already done so, please check out Praxis #6 Blog Carnival (my previous entry).

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a database of cell signaling

If you study cell signaling, here is a comprehensive database of cell signaling offered through the American Association for the Advancement of Science and Science Magazine: 

http://stke.sciencemag.org/cgi/collection

Components of cellular signaling pathways and their relations to one another–organized into pathways called Connections Maps–serve as the graphical interface into the database.  This information is provided for this databased by scientists with expertise in a given field, designated as Pathway Authorities, and includes canonical or general data about cell signaling, as well as specific data about particular signaling processes in specific organisms and cells.

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phylogenetics resource

If any of you are interested in phylogeny, here is a link to an amazing collection of programs and software for phylogenetic analysis. I have also included this link in my “research resources” sidebar.  It is hosted at the University of Washington, which has a track record for being powerhouse in phylogenetics. Programs included here include the standards of the field, which include PHYLIP, PAUP, TREEVIEW, etc. as well as many others.

There are in fact close to 400 software packages on this website (most of which are freeware) and are categorized as: general purpose programs, parsimony programs, distance matrix methods, computation of distances methods, quartets methods, Maximum likelihood and Bayesian methods, Artificial-intelligence and genetic algorithms methods, Invariants (or Evolutionary Parsimony) methods, Interactive tree manipulation, Looking for hybridization or recombination events, Bootstrapping and other measures of support, Compatibility analysis, Consensus trees, subtrees, supertrees, and distances between trees, Tree-based sequence alignment, Gene duplication and genomic analysis, Biogeographic analysis and host-parasite comparison, Comparative method analysis, Simulation of trees or data, Examination of shapes of trees, Clocks dating and stratigraphy, Model Selection, Description or prediction of data from trees, Tree plotting/drawing, Sequence management/job submission, Teaching about phylogenies.

If your research requires phylogenetic analysis tools, check out this website!

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new product: miltenyi macsquant analyzer

For those of you to whom this might be useful, the company Miltenyi Biotec describes the MACSQuant analyzer as a novel compact benchtop cell analyzer for highly sensitive multicolor flow analysis with the following attributes: 

  • Compact benchtop design
  • Multiparameter cell analysis
  • Absolute cell counting
  • Sensitive rare cell analysis using MACS® Technology
  • Multisample processing
  • Autolabeling of samples

I’ve never used this device before but have previously used other devices and reagents from Miltenyi with good results and support.  Anyway, this sounded interesting to me so I thought I’d pass it on.

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intragenic microRNA scanner

For those of you who do work on miRNAs, here is another resource that you can use to look for intragenic miRNAs.  You can either enter a miRNA ID and look for which gene it is located in or search with a gene name, gene symbol or ENTREZ gene ID to look for miRNAs within that gene:

http://intragenic.astridresearch.com/intragenic/login

As a reminder, MicroInspector is another miRNA research resource that I have written about before on this blog.

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nash equilibrium article

You may have seen the movie “A Beautiful Mind” and heard about John Nash, the Princeton mathematician who won the Nobel prize in economics for his work on mathematically describing what has now been termed “Nash Equilibrium,” which can be described as (excerpted from Wikipedia):

a solution concept of a game involving two or more players, in which each player is assumed to know the equilibrium strategies of the other players, and no player has anything to gain by changing only his or her own strategy (i.e., by changing unilaterally). If each player has chosen a strategy and no player can benefit by changing his or her strategy while the other players keep theirs unchanged, then the current set of strategy choices and the corresponding payoffs constitute a Nash equilibrium. In other words, to be in a Nash equilibrium, each player must answer negatively to the question: “Knowing the strategies of the other players, and treating the strategies of the other players as set in stone, can I benefit by changing my strategy?”

Stated simply, Amy and Bill are in Nash equilibrium if Amy is making the best decision she can, taking into account Bill’s decision, and Bill is making the best decision he can, taking into account Amy’s decision. Likewise, many players are in Nash equilibrium if each one is making the best decision that they can, taking into account the decisions of the others.

If you have ever been curious to check it out, here’s the original paper by John Nash that described this Nobel-prize winning concept: 

jr-nash-pnas-1949

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non-parametric statistical tests

Nonparametric tests are also referred to as distribution-free tests. These tests are used when you cannot make the assumption of normality. Non-parametric tests compare medians rather than means and, as a result, if the data have one or two outliers, their influence is negated. But how do non-parametric tests compare to the usual parametric statistical tests? Here’s a table:

Parametric test

Non-parametric analogue

One-sample t-test

Nothing quite comparable

Paired sample t-test

Wilcoxon T Test

Independent samples t-test

Mann-Whitney U Test

Pearson’s correlation

Spearman’s correlation

(this table is as much a reminder for me as everyone else!!!)

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how notch heals a broken heart

From the December 22nd issue of the Journal of Experimental Medicine:

The Notch pathway helps a developing heart get into shape but also helps a damaged heart reshape itself and keep pumping. Weakened by a heart attack or the protracted stress of hypertension, the heart rebuilds itself as it struggles to maintain blood flow.  Some studies suggest that heart stem cells begin proliferating to replace lost cardiomyocytes. Because the Notch pathway helps control self-renewal by other organs, it was hypothesized that it might also have a hand in heart remodeling.

In this study by Croquelois et al, when Notch1 was absent, mice with stressed hearts showed signs of over-exuberant repair including thicker ventricular walls and increased fibrosis–all signs of heart failure that are seen in humans after heart damage, for example after a heart attack.  These mice and their heart muscle cells also had a higher-than-normal death rate. Loss of Notch also spurred more heart stem cells to differentiate. Notch might allow the heart to conserve these cells.

The Notch pathway triggers stem cell proliferation in skeletal muscle, and the pathway short-circuits as we age. The researchers say that it’s possible the same deterioration occurs in the older heart, explaining why elderly people are more vulnerable to heart failure.

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il-12 and il-23 inhibitor shows promise for severe psoriasis

Ustekinumab is a human monoclonal antibody that antagonizes signalling of the heterodimeric cytokines interleukin 12 (IL-12) and IL-23 by binding to their shared p40 subunit. Previous work has suggested that IL-12 and IL-23 might be particularly important within psoriatic plaques (overexpression in both mice and humans with genetic deficiencies in these genes significantly decreased atopic responses). Phase III trials of ustekinumab have been very promising, with ustekinumab demonstrating significantly better results than etanercept (Enbrel), a soluble TNF-alpha receptor and currently one of the most effective treatments for severe psoriasis. For more information on the phase III clinical trials of ustekinumab, check out these updates:

http://www.nature.com/nrd/journal/v7/n11/full/nrd2753.html

http://www.nature.com/nbt/journal/v26/n12/pdf/nbt1208-1317a.pdf

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“super” cytotoxic t-lymphocyte clones for control of hiv infection

One of the hallmarks of HIV infection is rapid mutation and subsequent “escape” from adaptive immune responses–in particular the CD8+ cytotoxic T lymphocyte (CTL) response, which has been shown to be very important in the control of HIV replication in vivo.  Despite the ultimate failure of CTL responses to keep up with the escape mutations, CTL responses are still being investigated as a possible therapeutic tool.  A recent article by A Varela-Rohena et al in Nature Medicine has demonstrated the feasibility of generating “super” CTL responses with extremely high affinity to the HIV peptide/MHC-I complex that can not only recognize a cognate epitope but also epitopes with escape mutations. 

Why is this important?  Well, it gives re-newed hope for using vaccine-type strategies for using the immune system to overcome the barrier of genetic diversity and eradicate HIV infection.  Cool stuff!

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therapeutic interventions for endoplasmic reticulum stress

The endoplasmic reticulum (ER) is a subcellular organelle that is responsible for numerous functions including calcium homeostasis, protein secretion and lipid biosynthesis. However, cell death signaling pathways can be activated by prolonged stress to the ER originating from sources ranging from hypoxia, oxidative injury, high-fat diet, hypoglycaemia, protein inclusion bodies and viral infection. In fact, a number of disease processes are mediated and propagated through ER stress that lead to initiation of apoptosis. As increasing insight has been gained into the molecular mechanisms regulating ER stress and subsequent cell death, the potential for therapeutic benefit has been realized. 

Why is this cool?  ER-specific processes have been found to contribute or cause many diseases including Alzheimers, prion disease, Parkinson’s, heart disease, diabetes, cancer and autoimmune diseases.  The ER is currently a novel therapeutic target and represents an avenue for considerable future impact.  And as drugs are now being developed to target ER stress, we will see a lot of diseases being treated much more effectively than now.  For more information, here is a review of molecular mechanisms for handling ER stress and subsequent activation of apoptosis as well as the clinical implications of this information.

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t cell homeostasis – more interesting than it sounds

Look–we all know that T cells are important right?  I mean, it happens when someone is infected with HIV and loses just the subset of CD4+ T cells: AIDS.  There you have it–case closed. 

Okay, so in a healthy person, where do T cells come from?  Easy–the thymus.  But as we grow older, the thymus becomes involuted, decreases in size and eventually ceases production of  new T cells.  It is not completely known where new T cells come from in older age but it is believed that there is some production in the bone marrow, however at much lower rate than by the thymus.  So why is it then the number of T cells we have doesn’t significantly decrease we as grow older? 

You see!  The question of T cell homeostasis is actually quite interesting!  If I have piqued your interest in the least, check out this excellent review published just 4 days ago on the latest and most up-to-date information we have on T cell homeostasis:

Charles D. Surh and Jonathan Sprent, Immunity 2008

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PhosphoPep – a database of protein phosphorylation data

PhosphoPep is a database of mass spectometry-derived phosphorylation data currently from 4 different organisms: the fly (Drosophila melanogaster), human (Homo sapiens), worm (Caenorhabditis elegans), and yeast (Saccharomyces cerevisiae).

PhosphoPep offers different software tools which allow users to browse through single proteins, through pathways, and importantly to integrate the data with information from external sources, like protein-protein interaction data. Finally all data can be readily exported e.g. for a targeted proteomics approach and the generated data can be again validated using PhosphoPep, enabling systems biology signaling research.

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antibody resources for experiments

Good antibodies are critical for any research involving qualitative or quantitative protein detection, isolation, localization and visualization (e.g. western blots, immunoprecipitation or immunohistochemistry). Good antibodies that specifically bind to your protein, at a specific epitope—reliably—can make your life and research so much easier. In contrast, if your antibody is non-specific, you will pick-up a ton of non-specific signal from other proteins, which may (depending on the application) significantly confound your results. Obviously, controls are a must and can be very helpful in interpreting your results in the setting of noise, but antibodies can sometimes generate so much noise that your desired signal is drowned out.

Usually, you will be able to get good antibodies for well known and well characterized proteins from most companies but as you start looking more obscure proteins, it gets harder and harder to find good antibodies as well as a good variety in the epitopes to which the antibodies are directed. There was a time in graduate school when I was looking at some rather obscure proteins—you know they are obscure when they are called [cytokine]-stimulated gene-1, [cytokine]-stimulated gene-2, [cytokine]-stimulated gene-3, etc.—and a simple western blot became a brutal task. A lot of times, you will have to go to specific labs that have generated antibodies to your protein (and this will be noted in the literature), but if you want to look for commercially available antibodies first, here are some companies that I, and other graduate students whom I have talked to over the years, have found to be pretty reliable in terms of quality as well as customer support (which I often didn’t take advantage of, but when I did was very surprised at how helpful they could be).

Abcam

Cell Signaling Technologies(for cell signaling, in particular anti-phospho-protein antibodies)

Santa Cruz Biotechnology (a lot of variety—but reputation for quality is hit or miss amongst people I’ve talked to)

BD Biosciences (*the* place for conjugated flow cytometry antibodies)

Jackson ImmunoResearch Laboratories (for secondary antibodies)

These are of course just places that I’ve had routinely good experiences with. However, sometimes you may also find much smaller companies with very specific research focuses marketing very good antibodies to your protein, so try Google as well if you are trying to find antibodies to a particularly obscure protein or protein family.

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little magnetic beads saved my phd

One mainstay method for cell sorting is with flow cytometry. However, this can be a time intensive procedure and depends on the availability of a flow cytometer with sorting capability and someone who can run the machine. And while most major universities have flow cytometry cores with dedicated technicians, who often know waaaayyy too much about flow cytometry, you will have to schedule an appointment along with everyone else in the university who needs cell sorting.  At the institution where I did my PhD research, there were several sorting facilities but only one of them could handle my samples and the technician who ran that facility worked from 10am to 3:30pm (what is this, a bank?!?!?) performing only two sorting runs per day.  And with the number of people who wanted to use that sorting facility, I was left having to schedule my cell sorting 1 month ahead of time. The problem I ran into was that most of my experiments were dependent on getting purified cell populations from blood samples and when I could get blood samples was quite unpredictable. Plus on top of that, the sorting tech wouldn’t let me schedule more than 2 sorting runs at a time. At the rate I was going, I would be able to do maybe 1 experiment per month and my PhD would take on the order 10 years to finish. Painful. Just painful.

brilliant!

brilliant!


But then one of the older mudphudders to whom I was venting turned me onto cell sorting using magnetic beads and it saved my thesis project. Magnetic beads, attached to antibodies specific for a particular cell surface marker are used to label all cells with that marker and a magnet is then used to pull those cells out of an otherwise heterogeneous population of cells. Brilliant! All of a sudden, my cell sorting needs were no longer dependent on the availability of anyone but myself and I could do my sorting whenever I needed to—day or night. I will point out for anyone interested that sorting with magnetic beads: (1) is probably best characterized for isolation of specific cell populations from blood samples although can be used well with cell suspensions isolated from a tissues; (2) may be toxic to cells, depending on the cell type and the company you buy your beads from (beads from different companies–e.g. Miltenyi or Dynal–are made of different components); (3) will stay on the cell’s surface a lot longer than antibodies used for flow cytometry and may block any receptor/ligand interactions involving the cell surface protein in future experiments with those cells; and (4) usually does not allow sorting for the presence of more than one cell surface marker (i.e. you can only isolate cells that are positive for marker X *and/or* marker Y not cells positive for marker X *and* marker Y), unlike flow cytometric cell sorting—there are exceptions to this, in particular any bead that can be removed from the cells after sorting. Using this technology, I was able to get specific cell populations (sorting on 2 markers, actually) at purities and yields comparable to flow cytometric sorting (after some optimization of course).

When I was starting my PhD, cell sorting with magnetic beads wasn’t nearly as well known as it is today (…definitely aging myself…) so probably everyone reading this is already aware of that technology. but I wanted to mention this story because anytime you are dependent on a core facility for a necessary part of your research but are getting held up by logistics—odds are that there is technology out there that can let you get around it. I think the most frustrating thing is when you want to do work but can’t—and the worst is logistical problems, like when you are working 12 hours per day and you are dealing with a tech who works 5-6 hrs/day (in all fairness, I will say that the techs at another sorting facility that couldn’t handle my sorts were extremely helpful and really taught me everything I know about flow cytometry). You just gotta keep talking to people—graduate students, postdocs and PIs—and looking at product catalogues. If you are getting jammed up by logistics, odds are that many others have too and so some company has probably developed some solution that you need to find.

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DT40 cell culture media recipe

DT40s are a cell culture line of chicken lymphocyte B cells.  They exhibit a high rate of targeted (and random) integration of transfected DNA constructs and are ideal for stable transfection.  They can, however, be a royal pain to culture.  Anyone who has worked with DT40s can attest to the fact that it is probably the most finicky cell line they have ever worked with.  The key to keeping the DT40s alive and thriving is knowing how often to split them (i.e. not letting them get too dense while not splitting them too early…80% plate density is just about right) and knowing what environment (i.e. cell culture medium) to grow them in.  Through trial and error, a good friend of mine used the following culture media recipe for both her wild-type and stably transfected DT40s (for experiments that eventually got published in Nature Cell Biology)

To make 1 Liter:

RPMI 1640 Medium (w/ sodium bicarb & w/o glutamine) - 875 mL

1 microMolar glutamine - 5 mL

10% fetal bovine serum - 100 mL

1% chicken serum - 10 mL

50 microMolar 2-mercaptoethanol - 3.5 microliters

Penn/Strep (0.5 mg/mL G418) - 10 mL

Sterile filter.

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grants, scholarships, fellowships

There are now over 100 grants, scholarships, fellowships for research (or just for you!) for medical students, graduate students, postdocs, residents, clinical fellows and investigators now on my funding opportunities page.  On the way to two hundred!

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game on

Okay, funding opportunities are searchable now. 

It’s a work in progress!

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western blotting reagent recipes

All reagents for western blot are mostly purchased by labs, but if you still make your own reagents, here are some recipes I used during graduate school. These reagents are compatible with all pre-made gels. If you really want a recipe for SDS protein gels, let me know…

10x Transfer Buffer (1L):
144g Glycine
30g Tris

10x TBS (2L):
48.4g Tris Base 7.7
160g NaCl
Fill to 2L with dH20
pH to 7.6

Blocking buffer:
1 part 10x TBS
9 parts dH2O
5% mass/vol non-fat powdered milk
0.1% vol/vol Tween20

Wash Buffer:
1 part 10x TBS
9 parts dH20
0.5% Tween20

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where to buy sodium borate electrophoresis buffer

There was some interest in the sodium borate buffer DNA electrophoresis so I thought that I would look around for companies that sell the stuff.  I actually had a hard time finding anyone who sells sodium borate electrophoresis buffer (e.g. couldn’t find it in Invitrogen, Biorad, etc.), but the company Labsupplymall does sell a 20X stock of sodium borate buffer for nucleic acid electrophoresis–in case you don’t feel like making it yourself…

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sodium borate nucleic acid electrophoresis buffer

Sodium Borate Electrophoresis Buffer (SBE buffer) is an excellent alternative to traditional TBE (TRIS borate-EDTA) and TAE (TRIS acetate-EDTA) electrophoresis buffers. SBE buffer allows for higher voltage runs, extremely rapid running times, and high-resolution band separation.

Brody and Kern described sodium borate buffer for nucleic acid electrophoresis several years ago (Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis, Biotechniques 36, 214-216, Feb, 2004).

FYI – sodium borate buffers for gel electrophoresis are compatible with gel purification and most other applications.

You can buy pre-made sodium borate stock solutions or you can make it yourself. There is a recipe for 20X stock in the Brody and Kern article or here is one I found online:

for Borate Buffer: pH 7.4-9.2
Borax (sodium tetraborate) 0.2M = 76.2 gm/L
Na2B407*120H20 (MW = 381.37)
Boric acid 0.2M = 12.37 gm/L
H3BO3 (MW = 61.83)
Add boric acid to borax solution until desired pH is reached. Dilute to desired molarity with ddH20

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