Anti-Hsp90 antibody (ab13495)

AbID: 13495, Anti-Hsp90 antibody Rating: Below Average Image: Anti-Hsp90 antibody for Immunocytochemistry/ Immunofluorescence (Mouse) Sample: Species: Mouse Type: Cell Specification: neuronal Application: Application: Fixative: Methanol Permeabilization: Yes Blocking step: BSA as blocking agent for 30 mins at rt°C Other detail: Dilution: 1/100 Incubation time: 14 hours at 4°C Diluent: PBS + 1% BSA Secondary Antibody: Name: Non-Abcam Antibody was used: Rabbit alexa 488 Conjugation: Alexa Fluor® 488 Dilution: 1/100 Additional Data:

ANSWER:

Thank you for submitting your Abreview and for your honest feedback.
As you had rated your experience in the review as 'Below average', I just wanted to follow up with you to get some more details as to what you were unhappy about regarding use of ab62817 in your ICC experiiment.
Thanks in advance for the additional information.

Dear Sirs I have your product Anti-Hsp90 antibody (ab13495) and I want to immunoprecipitate hsp90 but I cant find the concentration of the antibody. Can you please feedback me on this? Best Regards  

ANSWER:

Thank you for contacting us with your question about ab13495. As this Hsp90 antibody has not been purified and is provided as whole antiserum, we do not know the specific IgG concentration. We estimate that the specific antibody concentration in whole antiserum is less than 0.5 mg/mL. For IP, we recommend using whole antiserum antibodies at a dilution between 1:50 and 1:100 in IP, so you may want to try a couple dilutions within this range in order to optimize the dilution to your samples. I hope this information will be useful, but please let me know if you have any further questions and I'll be happy to help.

Thanks for your answer. I attach the file for WB protocol. Could you read it and tell me what the fault is? I'm looking forward to hearing you. Thanks.

Western Blot

A) 1-DE gel electrophoresis - gel : NuPAGE, T10% (1.0 mm x 10 wells) (InVitrogen product) - sample amount : I can’t figure out. It must be below mg range. - sample was heated @ 95 degree for 5 min with mercaptoethanol.

B) WB - transfer proteins to the membrane for 2hr in a cold room. - rinse the membrane with blocking buffer for 1 hr in a cold room. - add 1/10,000 diluted HSP90 antibody (diluted with blocking buffer/Tween 20) to the membrane and shake it for 2 hr at room temperature. - rinse the membrane with PBST for 30 min. - incubate the membrane with secondary antibody (Alex 680 goat antibody raised in rabbit IgG, diluted with blocking buffer/Tween 20) for 1 hr at room temperature in dark condition. - rinse the membrane with PBST for 30 min. - rinse the membrane with deionized water for 30 min. - scan the membrane by Odyssey.

ANSWER:

Thank you for sending details regarding your protocol. At this point i would like to make the following suggestions. Try incubating with the primary antibody for overnight at 4C. Ensure that the protein transfered properly to the membrane (you can check this with Ponceaue S). Also, you mentioned that your secondary antibody was "goat antibody raised in rabbit IgG." As this primary antibody was raised in a rabbit, the secondary antibody must be anti-rabbit IgG. For example, goat polyclonal to rabbit IgG (raised in a goat). Please check to make sure that your secondary antibody is compatible for use with ab13495.

I hope this helps. Please contact us again if you need additional assistance with this antibody.

Can you confirm that the recommended dilution factor for ab13495 is 1/35000?

ANSWER:

Thank you for your email. Yes, it is recommended to use ab13495 at a dilution of 1:35,000 in Western blotting. This dilution was found to be sufficient to detect Hsp90 in HeLa cell lysate. If you have any further questions, please let us know.