Open Forum on Methodology


Open Forum on Methodology

Antibodies to phosphorylated MAP kinase

Nov 8 2000 12:05PM

Eve De Lamirande

I recently bought antibodies (a mono- and a polyclonal) against activated MAP kinase (pMAPK). These antibodies were raised against doubly phosphorylated TEY (*TEY*) of the active center of MAPK. I observed that the antibodies recognize many proteins beside MAP kinase and that interesting things are happening with these other proteins at the time of activation/inhibition of signal transduction. This labelling does not appear unspecific as it changes with treatments and time course of cell activation.

Would anybody know of other proteins, beside MAP kinase, that have the *TEY* motif when they are activated?

Collagen-induced signals in Entamoeba

Nov 20 2000 7:13AM

Bernard Rajeev

I want to study the collagen-induced signal transduction pathways occuring in Entamoeba histolytica. How can I study this? What are the best methods which I should use?

Histone Preps

Nov 22 2000 8:27AM

Jabed Iqbal

I am trying to isolate histones from asynchronous yeast cultures. When I run extracts on SDS-PAGE gels, some do not contain all 4 histone bands (2A,2B,3 and 4), whereas others do. How do I solve this problem?

Purifying pombe cyclins

Nov 30 2000 7:34AM

Stuart Taylor

Does anyone know a simple effective way to isolate and purify cyclins and CDK's from S. pombe; are antibodies available to Cyclins from pombe?

Stu Taylor

Isolation of metalloproteinases from various tissue sources

Dec 15 2000 9:56AM

M. A. Sciamanna

Would like an easy prep for isolating MMPs from various tissue sources, fresh and frozen. What needs to be done with tissue prior to W. Blot analysis?


Dec 15 2000 9:48AM

Joe Brozinick

We have been doing GSK3B IP kinase assays from intact muscle tissue lysates. These lysates are made from muscles that have been exposed to insulin either by in vivo injection of insulin (30 U/kg BW for 10 min) or by in vitro incubation with 133 nM insulin for 20 min. We have been using the GSK3B monoclonal antibody from Transduction labs. The assay was working very well, but the in the last several months we have been detecting an increase in activity with insulin, rather than a decrease. We have tried different assay buffers, different homogenization buffers and different substrate peptides (CREB,EIF2, and GS specific) but have gotten the same results. Does anyone have any suggestions?

How to do annexin V staining?

Dec 15 2000 9:55AM

Chuan-Wei Jang

For detecting apoptosis, I tried annexin V staining. But after modifying reagent concentrations, temperature, and incubation time, I could not get the expected result! My experimental cell lines include Hepatoma 3B, 293T, MCF10A etc, but none seems to work. When I carried out DNA staining and FACS analysis, I can see many sub-G1 cells, but with the same sample, I see no annexin V-positive signal or get irrelevant results. I followed the usual method, harvested cells with trypsin, then wash twice, and stained. I also have asked others who used this method and they told that is a tricky!! Is it true? Are there key problems to be avoided? Thanks for your help.

C.W. Jang

Elution of GST fusion proteins

Jan 16 2001 6:48AM

Mary Harte

We have a GST fusion protein that we are not able to elute off of the glutathione agarose using 20mM glutathione in 50mM tris -HCl pH8. Does anyone have any suggestions for eluting this fusion protein from the beads? Thanks, Mary Harte

Add salt

Jun 19 2001 6:49AM

John Ransom

Try to include 0.5-1 M NaCl in the elution buffer. It worked for some proteins (e.g. GST-ATF2)

Western blot detection of GPCR:GFP fusion proteins

Jan 16 2001 6:52AM

Jay Slack

We have been expressing several GPCR:GFP fusion constructs in HEK293 cells and have been unable to confirm expression of the appropriate fusion protein. Cellular distributions differ for each of the constructs, suggesting that the correct protein is being made. However, we need to confirm that the correct fusion protein is made before proceeding with functional analyses. Any suggestions/input regarding preparation of lysates, cell numbers needed/evidence of lack of reactivity of anti-GFP antibodies to GPCR:GFP fusions would be greatly appreciated.


Jay Slack

GPCR-GFP fusion detection

Jan 17 2001 2:04PM

Nadik Abdulaev

See "Constitutive arrestin-mediated desensitization of a human vasopressin receptor mutant associated with nephrogenic diabetes insipidus" by Barak et al. in Proc. Natl. Acad. Sci. USA January 2, 2001. Volume 98(1),93-98 for help. Good luck.

Measurement of NIK Activity

Jan 26 2001 11:46AM

Ramesh Natarajan

I am looking for a reliable antibody for NFkB Inducing Kinase(NIK) for immuno-precipitation and subsequent activity measurement. Thanx.

serine-threonine antibody

May 3 2001 1:03PM

Alex Lyakhovich

Does anybody know of any commercialy available serine-threonine antibody (total or phospho)? Thanks.

Peptide substrates for protein kinases

Jul 5 2001 10:51AM

Jerlyn Beltman

I am planning to synthesize some peptides to be used as substrates in an in vitro protein kinase reaction. Does anyone know the minimum length that can be used? I would like to use the shortest possible peptide.


Aug 6 2001 12:41PM

Phillip Abbosh

We are trying to perform chromatin immunoprecipitation on mamammalian tisue cultured cells. We are not getting much DNA yield so I would like to see what kinds of yields people typically get, and if there is something we are missing that might help.



RNAi controls

Mar 12 2003 12:59PM

Vicky L Emuss

Does anyone know where/how I can obtain single-stranded RNA oligos to use as negative controls for RNAi experiments.

Thank you!

RNAi controls

Mar 31 2003 7:12AM

Taosheng huang

I am looking for the same information. I called a few companies, but they were not willing to give out the information. I will let you know if I have an anwser. Would you please do the same if you have information first? Thanks,


RNAi controls

Mar 31 2003 7:26AM

Nancy R. Gough

Two STKE Protocols describe methods for the application of RNAi. The subject of RNAi controls is addressed in the STKE protocol by Lassus et al. In addition, Worby et al. describe the application of RNAi to cultured Drosophila cells. PDF versions of these Protocols are available with a site license, individual subscription, or by using the "Order this article" link in the left navigation area from the abstract view of the article.

P. Lassus, J. Rodriguez, Y. Lazebnik, Confirming Specificity of RNAi in Mammalian Cells. Sci. STKE 2002, pl13 (2002).

C. A. Worby, N. Simonson-Leff, J. E. Dixon, RNA Interference of Gene Expression (RNAi) in Cultured Drosophila Cells. Sci. STKE 2001, pl1 (2001).


Jul 18 2003 10:02AM

Gavin J Clydesdale

Dear Forum Members

From a non-anonymous Gavin Clydesdale

I am having trouble establishing a reliable method to stimulate detectable levels of phos-c-Jun in Jurkat cells.

I am after a reliable method of stimulation that will cause normal (non-transfected)Jurkat cells to produce detectable amounts of phosphorylated c-Jun via western blotting ( and flow cytometry would also be helpful).

The details of the stimulating agent plus the optimal time point at which to assess the phosphorylation are important. References would also be helpful.

Thank you

Kind regards

Gavin Clydesdale (

Flow cytometry protocols

Jul 18 2003 10:18AM

Nancy R. Gough

Dear Gavin,

Science's STKE has two protocols relating to flow cytometry that might be useful to you.

K. Grebe, T. Potter, Enumeration, Phenotyping, and Identification of Activation Events in Conjugates Between T cells and Antigen-Presenting Cells by Flow Cytometry. Sci. STKE 2002, pl14 (2002). [Abstract] [Full Text]

T. Zell, M. K. Jenkins, Flow cytometric analysis of T cell receptor signal transduction. Sci. STKE 2002, pl5 (2002). [Abstract] [Full Text]


Nancy R. Gough, Ph.D.
Managing Editor, Science's STKE

Antibody for HMGA1(HMGIY)

Jan 4 2005 6:43AM

sebastien jauliac

We are looking for an antibody that recognizes the human HMGA1 (HMGIY) protein on whole cell lysate, but we have huge difficulties finding one. If somebody knows one that works, it will be great.

Thank you

Cell fractionation

Jul 12 2005 6:12AM

Bakary Sylla

July 11, 2005

Question on cell fractionation:

Does anyone have a good protocol (or suggestion, or explanation) for cell fractionation in order to separate nuclear from cytoplasmic fractions? We are trying to show that a protein that shows a clear nuclear localization in immunofluorescence assay can be found predominantly in the nuclear fraction. However, using the cell fractionation protocol we have, the protein shows equal distribution between cytoplasm and nucleus, whereas the control proteins (tubulin and PARP) show the expected distribution. We think that probably our protein is loosely present in the nucleus and becomes easily leaky during the process.

Thank you for your help and suggestions.



Protocol being used:

  • Cells are resuspended in buffer A (10mM HEPES, pH 7.9, 1,5 mM MgCl2, 10 mM KCl, 10% Gycerol, 340 mM sucrose) supplemented with protease inhibitors.
  • Add Triton X-100 to 0.1%
  • Leave on ice for 5 min
  • Spin at 1300g for 4 min at 4 degrees
  • Transfer the supernatant to a new tube (cytoplasmic fraction)
  • Wash the pellet (nuclei) with buffer A and add 1XSDS- PAGE buffer, sonicate to obtain the nuclear fraction

    Cell fractionation - response

    Jul 21 2005 3:31PM

    Larry Leung

    I recommend using the protocol published by N. C. Andrews and D. V. Faller [Nucleic Acids Research 1991 vol 19 p. 2499 (PubMed Central)].

    The article can be downloaded free from PubMed Central.

    Best of luck.