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BATCH NUMBER 97820 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Multiple bands- I get about 7 bands in size 20-47 kDa; most of the bands are biger and one band are smaller than 23 kDA (-the size og TIMP3 protein). SAMPLE I used HEK cell line as a positive control and platsenta tissue as a sample- both of them gave lot of non-specific bands(-the bands were on the same size). PRIMARY ANTIBODY I used the dilution of 1:500. Manufactor abcam. Incubated ouvernight 4 C. Washing 2,5% of w/v nonfat dry milk in TBST 3X10min. ANTIBODY STORAGE CONDITIONS -20 C SAMPLE PREPARATION SDS- Loading sample buffer (with DTT). AMOUNT OF PROTEIN LOADED Control gel was stained with commassive- the amount of the protein was OK. ELECTROPHORESIS/GEL CONDITIONS 12% of Gel; reducing TRANSFER AND BLOCKING CONDITIONS Semidry 1 hour; buffer 24 mM Tris, 192 mM glycin, 20% methanol. Blocking agent 5% of w/v nonfat dry milk in TBST. SECONDARY ANTIBODY Anti- rabit. Manufacturer [ a competitor]. Dilution 1:10 000. Incubation 1 h. Wash step 2,5% of w/v nonfat dry milk in TBST 3X10min. 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? Primary antibody- dilution? I did western with TIMP1 and TIMP4 in same contitious and everything was OK. ADDITIONAL NOTES I did western with TIMP1 and TIMP4 in same contitious and everything was OK.
Asked on Oct 10 2005
I'm sorry to hear you are having a problem with aab2169 in WB. We have not tested this antibody in western blotting and it is possible that the antibody does not work in this application, or that it requires the protein to be in its native form. I would therefore like to suggest to run your samples in non-reducing (and maybe also non denaturing) conditions to test this possibility. You mention HEK cells as positive control and placenta tissue; 293 cell lysate and the choriocarcinoma cell lines JAR, JEG-3, B6 should contain high levels of the protein (according to Feng H et al, Gynecol Oncol. 2004 Aug;94(2):375-82) however I did not find a reference for normal placental tissue containing high levels of TIMP3. We have used human breast carcinoma tisseu as positive control for IHC. The lysis buffer used in the paper by Feng et al is SDS lysis buffer [50 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol], containing proteinase inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), is this the same as the one you used? If this is not similar the problem may also be due to an inadequate extraction of the protein. Finally, another possible problem may be the blocking buffer you are currently using. I would like to suggest blocking the membrane for 1hr in 5%BSA in TBST, then rinsing the membrane in TBST for 2 seconds and incubating ab2169 in TBST only. Between steps I would recommend washing in TBST only too and to incubate the secondary in TBST too. Please let me know if this helps and do not hesitate to contact us for further advice,
Answered on Oct 10 2005