Question (11380) | Rabbit Anti-Mouse IgG H&L (Alkaline Phosphatase) (ab6729)

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Question

>lot 52644 (My PO no: 04-12-0141) Abcam Reference No: 64641 (Invoice No: 48594) > > ab8226-200 > > >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? Rabbit muscle tissue >.Cell extract/ Nuclear extract? Tissue extract >.Purified protein? >.Recombinant protein? > >3. How much protein did you load? 10ug >.How did you prepare the lysate for the analysis (protease inhibitors etc)? sonicate with lysis buffer >.Did you heat the samples? yes > >4. Primary Antibody >.Specification (in which species was it raised against)? ab8226-200 >.At what dilution(s) have you tested this antibody? 1:250 >.Incubation time, wash step? 1 hr , wash - 5min, 15min, 5 min and 5 min > >5. Secondary Antibody >.Specification (in which species was it raised against)? ab 6729 >.At what dilution(s) have you tested this antibody? 1:5000 >.Incubation time, wash step? 1 hr, wash - 15min, 5 min 4 times >.Do you know whether the problems you are experiencing come from the secondary? No, other antibodies are within the specify range from supplier > >6. What detection method are you using? Perkin Elmer western Lightening chemiluminescence > >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? Yes (we use 0.3g skimmed milk, 5ul tween 20 in 50 ml TBS.) > > .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? Probably > >8. Optimization attempts >.How many times have you tried the Western? 4 times >.Do you obtain the same results every time e.g. are background bands always in the same place? I don't get any bands with the recommended dilution on the data specification sheet I get bands only when I use 1 :250 dilution. >.What steps have you altered? primary antibody dilution I have tried 1:10000, 1:1000, 1:500 and 1:250 (only the 1:250 dilution give bands) > >9. Did you apply positive and negative controls along with the samples? Please specify. No

Answer

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.

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