Question (11266) | Rabbit Anti-Mouse IgG H&L (Alkaline Phosphatase) (ab6729)

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Question

I have an enquiry which I don't know whether other of your customer faces it. I had bought ab8226 (Anti beta actin) a few weeks ago and according to the data sheet the recommended dilution for westtern blot is between 1/500-1/10000. I have tried with 1/10000 with no bands and 1/500 with super faint band after 1 hour exposure, then I tried with 1/250 and exposed for 1 hour and I get better result, the blot is attached. I am using Ab6729 (diltuon 1/5000) as secondary antibody.

Answer

Thanks for your email and I'm sorry to hear that you are experiencing difficulty with ab8226. To better assist you, can you please provide me with me with more details regarding your protocol? Also, please include the lot number that you received (it is located on the vial) and the Abcam order number or purchase order number that was used. Thank you, and I look forward to hearing from you. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). 2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein? 3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples? 4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? 5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary? 6. What detection method are you using? 7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •At what size are the bands migrating? Could they be degradation products of your target? •Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered? 9. Did you apply positive and negative controls along with the samples? Please specify.

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