Anti-Hsp90 alpha antibody [2G5.G3] (ab79849)

Thank you for yoru recent telephone enquiry.

I ampleased to let you know I now have the ELISA testing protocol for ab79849 -Hsp90 alpha antibodyfrom the originator. Thank you for your patience.
I have copied this below:

Purified hsp90alpha (1 μg) was coated on each well of a Maxisorp C96 titer plate (Nalge Nunc Int, Rochester, NY, USA) were incubated with various concentrations of purified mAbs in 0.1 mL of the blocking buffer (0.17 M H3BO4, pH 8.5, containing 0.12 M NaCl, 0.05% [vol/vol] Tween 20, 1 mM ethylenediaminetetraacetic acid, and 0.05% [wt/vol] NaN3 and 0.25% [wt/vol] bovine serum albumin) for 2 hours at room temperature.

After a 1-hour incubation with the anti-mouse Ig(G+A+M) and IgE conjugated to alkaline phosphatase (1/4000 dilution) and washing, the alkaline phosphatase activity was determined by measuring the absorbance at 655 nm after a 1-hour incubation with Bluephos microwell phosphatase substrate at 30°C.


Please note, this protocol would be a guideline only, and may require some individual optimization for individual experiments. I can also suggest to check the datasheet for ab79849 for further information. For example, it is tested and guaranteed for Mouse, Rat, Human. Which species will you be using?

https://www.abcam.com/Hsp90-alpha-antibody-2G5G3-ab79849.html (or use the following:
https://www.abcam.com/Hsp90-alpha-antibody-2G5G3-ab79849.html).

I hope this will be helpful. If you have any further questions, please do not hesitate to contact us.

Thank you for your reply. I will forward your request to 51AB, who will be in contact with you to process your refund request. I hope this information is helpful.  Please do not hesitate to contact us if you have any additional questions.

Thank you for your reply with this information. It seems that the lower band is certainly isoform 1 of HSP90 and it is likely that the higher band is isoform2. I am not an expert in this field and could not say for sure if both isoforms are expressed in your samples. If you are dissatisfied with these results, I am happy to replace the antibody, however it is possible you will see a similar result. Please do not hesitate to contact me if you have any additional questions.

Thank you for contacting Abcam regarding ab79849. I am sorry that you have been experiencing difficulties with this antibody in WB. I have reviewed the data provided and would like to point out that there is a second isoform of this protein that is predicted to be 98kDa and may be represented by this band. SwissProt: P07900 What are the blocking conditions that you tested? Also, have you tested other primary antibody dilutions? How long do you incubate with the primary antibody? I hope this information is helpful. I look forward to your reply so that I may assist you further. Please do not hesitate to contact me with additional questions.

Than you for your inquiry and I am sorry to hear that you are experiencing some trouble with ab53497 and ab79849. Are you running any isotype controls with these experiments? I just want to make sure that the pull down with the HBP21 antibody is genuine. Have you tried to do a regular IP and not a co-IP with ab53497 and ab79849? These are the IP references we know of with the HSP beta antibody, ab53497. Barent R. L. (1998) Mol. Endocrinol. 12: 342-354 11. Lo. M.A. (1998) EMBO J. 17: 6879-6887. What kind of lysis buffer are you using? A key factor in maintaining complex formation throughout the steps required for co-IP is the lysis and wash buffers. Many protein interactions will remain intact after lysis using standard non-denaturing lysis buffers. Buffers with low ionic strength (i.e., <120mM NaCl) that contain non-ionic detergents (NP-40 and Triton X-100) are less likely to disrupt protein-protein interactions; however, testing may be required to determine the best buffer formulation for a specific protein complex of interest. Additionally, lysing cells by sonication or vortexing the lysate or bead-bound immune complexes during the wash steps should be avoided to prevent the disruption of the protein-protein interaction(s) of the target complex. And while centrifugation is a standard method to separate the precipitated complexes from the remaining lysate and during wash steps, the samples should be handled gently to prevent the loss of bound complex proteins. An advanced technique to strengthen protein-protein interactions is by crosslinking the binding partners. Using this approach, all proteins within the active distance of the specific reagent in a cell lysate are covalently crosslinked, and the target protein can then be immunoprecipitated along with the other proteins in the complex without the risk of losing binding partners. Co-IPs can be tricky due to the nature of the interaction of the proteins, nonspecific binding to IP components and antibody contamination that may mask detection. We haven't tested ab53497 and ab79849 by co-IP so it's difficult to know if this may be the issue. Could you try the protocol recommendations or run a regular IP with the antibody? That could show that it is an issue with the protein binding more than the antibodies.