Anti-MMP16 antibody (ab5709)

BATCH NUMBER 164655 ORDER NUMBER 248346

DESCRIPTION OF THE PROBLEM No signal. Blot was completely blank. When stripped and reprobed for beta actin, clean bands at 42 KDa were seen with the blot.

SAMPLE Rat whole ovary lysates in RIPA buffer with protease inhibitors and PMSF. Lysates were centrifuged at 4 C at 13 rpm for 30 minutes to pellet cell membrane fraction before being aliquoted and stored at -80 C. Each aliquot thawed not more than 3 times before discarding.

PRIMARY ANTIBODY Abcam- ab5709/rabbit polyclonal/diluted in blocking buffer (wash buffer, i.e. TBS with Tween +5% non fat milk)/used 2, 1 and 0.5 mcg/ml/incubated overnight at 4 C. Blocked in block buffer above for 1 h at room temp after transfer, before primary antibody. Next day, washd 3X for 5 min each in wash buffer.

DETECTION METHOD Attempted detection with ECL plus per manufacturer(Amersham)'s directions. Exposed to radiograph for 30 secs, 1 min, 5 min and 45 min. No bands observed on blot - no background even.

POSITIVE AND NEGATIVE CONTROLS USED No positive or negative controls used. But when blot was stripped and reprobed for beta actin, clear bands at 42 KDa were seen.

ANTIBODY STORAGE CONDITIONS aliquoted into 10 mcl amounts and froze at -20 C upon receipt

SAMPLE PREPARATION Sample mixed with 2x reducing sample buffer (containing Tris.HCl, Glycerol, 1%SDS, 0.05% Bromophenol blue and beta mercaptoethanol) and heated for 10 minutes at 95 C and cooled on ice before loading on to a mini gel.

AMOUNT OF PROTEIN LOADED 20-25 mcg

ELECTROPHORESIS/GEL CONDITIONS Reducing gel, 8% acrylamide. Running buffer has Tis, Glycine and 0.1% SDS. Gel run at constant current of 40 mA (about 70 to 80 volts) for 1.5 hrs until loading dye reached bottom of gel

TRANSFER AND BLOCKING CONDITIONS Transfer buffer has Tris, Glycine and 0.01% SDS. Wet transfer at 60 V in cold buffer for 1.5 hrs.

SECONDARY ANTIBODY Santa Cruz/goat anti rabbit(200 mcg/ml)/diluted in block buffer 1:5000 (manufacturer recommended dilution 1:5000 to 1:20000)/incubated 1 h at room temp/ washed 3x for 5 min ea. in wash buffer.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I have tried 3 different concentrations of the primary antibody.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you are experiencing difficulties with this product ab5709 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered.

I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. If the suggestions do not prove to be helpful, would you please be so kind to confirm the following items in order to help me better understand the cause of the problem.

Regarding the no signal, I would suggest increasing the amount of protein samples. At Abcam, we recommend using around 20-40ug per well. You may want to try increasing it to 40ug or more.

Most customers who use milk as a blocking agent tend to have problems detecting bands. At Abcam, almost all of our products have been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. I would recommend trying 5% BSA, if you have not already done so. Also, cross reaction between the antibodies with the diluent could also cause the lack of signals. We would normally use 1% BSA to dilute the primary and secondary antibodies. You can also use PBST only.

Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working.

I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me.