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

Thank you for contacting us for technical support with ab6729. I think the problem you are experiencing is due to the fact that budding yeast expresses an endogenous form of alkaline phosphate (http://www.expasy.ch/uniprot/O60109) and by using a secondary antibody conjugated to AP this creates a background signal. I would therefore recommend using a HRP conjugated secondary antibody (and ECl + detection kit) to prevent this problem. Please do not hesitate to contact us again if you still experience problems with an HRP-conjugated secondary antibody,

I have heard back from the originator of this product and would like to pass along the following comments to you (possible resaons why the AP loss activity). a). Researcher diluted the product and stored at 4oC. THE PRODUCT HAS TO BE STORED UNDILUTED. b). The product was accidently frozen, resulting in reduced activity. c). The pipette used for sampling was contiminated, that may accelarate AP degredation. That often happens. If you touched the tips, they may have been contaminated with DNAse, proteases etc. Also AP should be stored in dark tubes. d). During storage, the liquid evaporated to the cap, causing the antibody to dry. e). The substrate was at the wrong pH, or went off, so reaction was not at optimal conditions. I just wanted to pass this onto to you, please let me know if you would like a replacement vial of ab6729.

Thank you for your email. I have contacted the originator of this antibody in order to see if they have any suggestions or have heard of any problems with the particular lot that you have received. I will not hear back until next week and I'm also going to be away next week. I will pass their comments onto you when I do hear. In the meantime, if you would like to try a replacement vial, please let me know and I or one of my colleagues will arrange that for you. Thank you again, and have a great weekend.

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab6729. Can you please tell me 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 for the details that you have provided. I'm surprised that you are unable to see a signal with ab8226 except for the faint signal detected with the dilution of 1:250. We have had some good feedback from customers who have successfully used the antibody at dilutions from 1:1000 to 1:5000. I can certainly send you a replacement vial of ab8226 free of charge. Please let me know if you are interested.

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.