Anti-Desmin antibody [RD301] (ab8976)

I'm sorry for the belated reply. To answer your question. Yes, the old tube lot 225987 is not working. I hope that answers all of questions.

ANSWER:

Thank you for clarifying this information. As you have had good results with the other lot I believe the problems is with the vial you received and I would be happy to offer you a replacement vial if the antibody was purchased in the last 120 days. If you could please provide your order details I can arrange a free order for you,

I look forward to hearing from you,

BATCH NUMBER 22587 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM we did immunhistos with two of your lots. lot number 25677 gave a very nice Z-disc and intercalated disc staining. we used ist 1:200. Lot number 22587 did not give any signal at all. Two different persons used it at dilution 1:100 and 1:200. SAMPLE Mouse heart, frozen sections. PRIMARY ANTIBODY Desmin (see above) DETECTION METHOD Fluorescence microscop POSITIVE AND NEGATIVE CONTROLS USED as negative control we used only the second antibody. As positive control we used a second antibody in order to colocalize it with desmin. That worked.ANTIBODY STORAGE CONDITIONS -20°C FIXATION OF SAMPLE we used both : actone (-20°C) and Paraformaldehyde. It did not work on either sections. The other lot worked with acetone fixation. BLOCKING CONDITIONS BSA 2% in PBS for 1 hour SECONDARY ANTIBODY FITC anti-mouse Vector

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2

HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes

DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? We used different dilution and fixation methods. But after finding out that lot number 25677 works we did not do more tests.

ADDITIONAL NOTES I could send you the pictures I have taken that have worked. In this case please just write me an email.

ANSWER:

Thank you for contacting us for technical support regarding your recent problem with a vial of ab8976. I'm sorry to hear you are experiencing problems with this vial and I would appreciate if you could please clarify which vial is not working for you. According to your orders you have received two tubes: lot 256477 is the most recent one (the one you said worked well) was ordered a month ago lot 225987 was ordered in December 2006 (the one you said did not work). Is this correct or is it the opposite happening i.e. the new vial is not working? Thank you very much for clarifying, I look forward to hearing from you to resolve this matter,

What dilutions of ab8976 have you tried?

1:500, 1:400, 1:300

Do you know that the secondary antibody is working properly?

Yes, we used them to detect alpha actin, GAPDH and some other antibodies in the same days/same membranes.

Also, did you ensure that the protein transferred properly to the membrane?

Yes, we detect GAPDH on the same membrane.

Thank you again, and i look forward to hearing from you.

ANSWER:

Thank you for your email. I would expect for you to detect desmin in your samples, smooth muscle cells, and you certainly have tried high enough concentrations of ab8976. How long did you incubate with the primary? I would suggest incubating overnight at 4C.

Other than that, your protocol looks fine to me. We have not had any other complaints regarding this product, and there is a possibilty that the vial you received had gone off during the shipping process. I can offer to send you a free of charge replacement vial of ab8976 or can offer you a credit note or refund. Please let me know which you would prefer.

BATCH NUMBER 124239 ORDER NUMBER 92612

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Smooth muscle cells (human and mouse), Mouse aorta tissue extract

DETECTION METHOD Pierce SuperSignal West Piko Chemiluminescent Substrate

POSITIVE AND NEGATIVE CONTROLS USED Control for secondary antibodies - used primary anti human/mouse GAPDH

ANTIBODY STORAGE CONDITIONS -80 C

SAMPLE PREPARATION RIPA + protease inhibitor coctail

AMOUNT OF PROTEIN LOADED 30 ug

ELECTROPHORESIS/GEL CONDITIONS Reduced 8% gel

TRANSFER AND BLOCKING CONDITIONS Tris/Glycin buffer + 10% Methanol Tris buffer + 5% Milk (no fat)

SECONDARY ANTIBODY Pierce, anti mouse, 1:10000, 1:5000, 1:4000

1,5 hour

Tris buffer + 5% Milk (no fat)

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

ANSWER:

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab8976. Can you please provide me with a few additional details: What dilutions of ab8976 have you tried? Do you know that the secondary antibody is working properly? Also, did you ensure that the protein transferred properly to the membrane?

Thank you again, and i look forward to hearing from you.