Anti-Hsp90 antibody [S88] (ab1429)

On your spec sheet for ab1429 is states that the immunogen was the full length native HSP90 protein (that is why I purchased it). So it should react with native protein!. I have tried reduced conditions on a western blot and get no banding. Do you haive any monoclonal to HSP90 (needs to react with rat) that we could replace it with??

ANSWER:

Thank you for your rapid reply.

I am very concerned that reduced conditions in western blotting did not give you bands, we have not received any other complaints about this antibody which is very popular.

Could you please clarify the incubation time you have tried? Have you tried a 2ug/ml concentration? The recommendation of 1ug/ml is for detection with more sensitive kits like Ecl+ and the Pierce SuperSignal kit so by increasing the concentration of antibody you should get a stronger signal.

I look forward to hearing from you regarding the incubation time,

BATCH NUMBER 151629 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Rat Liver or 9L glioma cells

PRIMARY ANTIBODY (ab1429) anti-HSP90 mouse monoclonal

DETECTION METHOD BCIP/NBT

POSITIVE AND NEGATIVE CONTROLS USED Purified rat liver HSP90 (positive control)

ANTIBODY STORAGE CONDITIONS Frozen

SAMPLE PREPARATION samples (Cystosol) by homogenising in 20mM sodium phosphate, 1mM EDTA and 10% glycerol and spun at 80,000 g for 1 hr to collect cytosol. (protease inhibiors added) We have then been doing anion exchange and using ab1429 on dot blots to detect the fractions we want.

AMOUNT OF PROTEIN LOADED Dot blot of fractions coming off our ion exchange

TRANSFER AND BLOCKING CONDITIONS using 5% skim milk in PBS

SECONDARY ANTIBODY [another company] goat anti-Mouse alk phos

HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? performed at the recommended Mab concentration of 1ug/ml. We get no signal on Native protein coming off our Ion exchange. Similar western blots on Native HSP90

ANSWER:

I'm very sorry to hear you are experiencing problems with ab1429 in dot blot and in western blot. We have not tested this antibody in dot blot or in native western blotting and therefore the problem may be due to the fact that the protein in your samples is in its native form. I would therefore recommend to try in denatured reduced conditions. You do not mention what incubation time you have tried, I would like to recommend to do an overnight incubation in Tris buffer containing 0.01% Tween20 and to try 2ug/ml of antibody.

I hope the above recommendation will help, please do not hesitate to contact me again if I can be of further help,

Thank you for the response. Our constructs have FLAG at the amino-terminal end of the protein - however, I think there may be a misunderstanding of what I was doing. A Flag-protein fusion was expressed, it was purified via its flag tag, and mass spec showed that several proteins (Hsp90, MBD3, etc.) copurified with it. So in my westerns, the protein samples do not contain Flag-tagged Hsp90 or MBD3, but rather the endogenous forms of those proteins which copurified with my Flag-tagged protein. Sorry for the confusion.

ANSWER:

Thank you for your reply, and your clarification.

There are a couple of suggestions I would like to make:

- I would recommend switching your blocking and primary incubation steps, i.e. blocking for 1 hour at room temperature and incubating with the primary antibody overnight at 4C.

- I wanted to double-check that you are not adding blocking agent to the primary antibody solution, as this can sometimes overblock the membrane.

- The dilutions on the datasheet are recommendations only. I would try ab1429 at 1:100 to see if this improves your results. In addition, ab3755 is provided as whole antiserum. Since it is a polyclonal, I would have tried this one at 1:500 to begin. If you see an increase in background with the increase of antibody concentration, you may wish to add a very small amount of blocking agent back to the primary antibody solution (to 0.5%).

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

I have another question to consult you. I notice from your datasheet, the size of ab1429 is 50 ug, and the form of antibody is liquid whose concentration is 0.2mg/ml, which means the package is about 250ul liquid,right? and the working dulution you recommend for IHC-Fr is about 10-20ug/ml,which means I can use ab1429 for about 50 tests(supposing working dilution is about 20ug/ml and i use 50ul per test)right? I am a greenhand in perfoming IHC, maybe you can give me some suggestions.

ANSWER:

That is correct, you should receive 250ul and the recommended working dilution is 10-20ug/ml. I suggest that you refer to the IHC protocol section on the Abcam homepage which may be useful for you. If you have any more questions, please contact us again.

My research is about how HSP90 influences the glucocorticoid receptor function. I would like to use the antibody to detect HSP90 in human PBMC(peripheral blood mononuclear cell) by immunocytochemistry/IC, and I would like to detect total HSP90(including alpha and beta, both in free and complexed form), and I don't hope to see the cross reaction with glucocorticoid receptor in human PBMC. Can i use your product ab1429 to perform mmunocytochemistry? (PBMC is adhere to slide by cytospin)

ANSWER:

Thank you for your enquiry. All the information that we have regarding ab1429 is located on the online datasheet. Ab1429 has been tested for application in Western blot, Immunoprecipitation, and Immunohistochemistry (Frozen Sections). If you have more questions, please contact us again.