Rabbit polyclonal Secondary Antibody to Mouse IgG - H&L (AP) (ab6729)

BATCH NUMBER 131853 ORDER NUMBER MBPO/023

DESCRIPTION OF THE PROBLEM No signal of the relavant antigen (I have tried two different primary: see below), many non-target bands.

SAMPLE Budding yeast cell extract (PCNA-Myc and non-tagged).

PRIMARY ANTIBODY Abcam ab56 anti-Myc (9E11), Lot 130523, 1000x to 5000x dilution, 1 hr.

or

Abcam ab29 anti-PCNA, Lot 119707, 1000x to 5000x dilution, 1 hr.

DETECTION METHOD CDP-star reagent from Perkin Elmer.

POSITIVE AND NEGATIVE CONTROLS USED For detection of Myc-tagged protein, I included non-taggad sample as negative control. Size difference in Myc-tagged and non-tagged samples must help to identify correct band. Same blot has worked fine with HRP-Anti Myc from Santa Cruz, enabled me to compare the result.

ANTIBODY STORAGE CONDITIONS In fridge (4 degree) w/o dilution.

SAMPLE PREPARATION Roche Complete as protease inhibitor. Boiled soon after addition of SDS.

AMOUNT OF PROTEIN LOADED up to 10 ug.

ELECTROPHORESIS/GEL CONDITIONS Reducing, mainly 6% and 12%.

TRANSFER AND BLOCKING CONDITIONS 5% milk in PBS-T as blocking.

SECONDARY ANTIBODY ab6729

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I tried several different dilution of both primary and secondary (x1000 to x5000). At 1000x dilution, backgorund was very high. At 3000x or 5000x dilution, background was reduced, but no target band. Similar pattern of background bands. I also tried "No Primay". It gave quite similar background bands.

ADDITIONAL NOTES I used ab6729 in combination with ab29 anti-PCNA and ab56 anti-Myc. Both gave essentially identical result (no target band, many background).

The same blot worked fine for HRP-anti Myc from Santa Cruz.

Also not tested in western, I believe that ab56 anti-Myc is OK, because I and a student have used it for ChIP and got nice results.

Taken together, I conclude that an6729 doesn't specifically recognize mouse IgG.

I prefer replacement to other antibody with good record, such as ab6790, rather than refunding.

ANSWER:

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,

The lot # is: 71496 and the order number: 68495

Thanks a lot.

ANSWER:

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.

The lot # is: 71496 and the order number: 68495

Thanks a lot.

ANSWER:

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.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM loss of activity of the alkaline phosphatase

SAMPLE amyloid fibers from a-beta: used anti-abeta antibody and than used the anti mouse IgG as secondary antibody

PRIMARY ANTIBODY Abcam ab 10146 dilution 1:6000

SECONDARY ANTIBODY abcam ab 6729 dilution 1:1000

DETECTION METHOD UV-absorption

POSITIVE AND NEGATIVE CONTROLS USED yes

SAMPLE PREPARATION coated plates with amyloid, incubated with anti abeta and than with anti mouse IgG

TRANSFER AND BLOCKING CONDITIONS BSA blocking

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 20 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes

WHAT STEPS HAVE YOU ALTERED? incubation times and concentrations

ADDITIONAL NOTES I tried the assay many times to find the right concentration, and eventually found it. the conentrations were in the described range for these antibodies. However suddenly the turnover rate of the alkaline phophatase came to almost a stand still within two weeks of using it. Normally is took 30 - 45 minutes to get the desired response. Now it takes 15-20 hours to get a comparable response, with the same time and concentrations used before. does the alkaline phosphase decompose after a certain period of time? I kept it at 4 degrees as was said in the data sheet.

ANSWER:

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?

>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.