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

>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