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I have now tested the anti-p28 antibody we recently obtained from abcam (ab6502) using the dilution and fixation conditions you suggest. The antibody really does not detect what it is meant to. On infected cells the anti-p28 antibody is intense but not specific (it does show more intense staining than an isotype matched control antibody but, as I said, it appears totally non-specific and stains all of the cell very intensely). Perhaps worse, is the extremely intense punctate nuclear fluorescence it shows on un-infected cells (which is not shown with an isotype matched control). Clearly, this antibody is not specific for HCMV infected cells and does not detect pp28 expression as detailed in your data sheet. I am happy to try another Lot number if you think there may be problems with the batch we have received. Alternatively, if you can put us in contact with anyone in the UK or Europe who has purchased this antibody, we would be happy to contact them regarding our problems. Failing that, we would appreciate a refund on the purchase cost of the antibody and be happy to send you back the antibody which we have.
Asked on Apr 27 2004
Thank you for your enquiry. We are very sorry that you are dissatisfied with this particular antibody. We take your complaint very seriously and certainly we can offer you either a new vial - free of charge or a refund. Please do let us know which you would prefer. However, we would like to investigate this matter further and would highly appreciate if you could provide us a detailed protocol. This background information would help us to decide if the antibody is really specific for detecting Cytomegalovirus pp28. If not, then we will unpublish the product and withdraw it from our catalogue. 1. Please describe the problem (high background, no staining etc). 2. On what material are you testing the antibody in IHC? • Species? • Cell line? • Tissue? 3. How did you fix the samples? • Ethanol, methanol • Acetone • Paraformaldehyde • Other 4. Did you apply antigen retrieval step? • Enzymatic method • Heat mediated technique • Other 5. How did you block the unspecific binding sites? 6. Primary antibody • Specification (in which species was it raised against)? • At what dilution(s) have you tested this antibody? • Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? 7. Secondary antibody • What secondary antibody are you using? • Specification (in which species was it raised against)? • At what dilution(s) have you tested this antibody? • Incubation, wash steps? • Do you know whether the problems you are experiencing come from the secondary? • What detection method are you using? 8. Background staining • Please provide an image of your staining 9. Which detection system did you use? 10. Did you apply positive and negative controls along with the samples? Please specify. 11. Optimization attempts • How many times have you tried the IHC? • Do you obtain the same results every time? • What steps have you altered? Thank you for your understanding and your co-operation. We are looking forward to hearing from you soon.
Answered on Apr 27 2004