Anti-TIMP3 antibody (ab2169)

Customer is interested in possibly using this antibody with mouse samples. Based on the immunogen sequence, what is the homology with mouse?

ANSWER:

Thank you for your enquiry. The immunogen sequence is proprietary and we have not determined the homology to mouse. We would suggest comparing the C-terminus regions of human and mouse TIMP3.

Please contact us again if you have any additional questions.

For ab2169 - is there more information regarding the immunogen? What is the sequence, or the aa range? Also, what is the concentration?

For ab11814 - has the antibody been tested in IHC-formalin fixed paraffin-embedded sections, frozen sections, or both?

ANSWER:

Thank you for your enquiry. For ab11814 I regret to inform you that the antibody has not yet been tested for application in IHC. The antibody has only so far been characterized for application in Western blotting and I have updated the online datasheet to reflect this information.

For ab2169, the immunogen used was a synthetic peptide from the C-terminus region of the human TIMP-3. The exact sequence is proprietary. The concentration of the current batch is 100 ug/ml.

If you have any additional questions, please contact us again.

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.

ANSWER:

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,

We want to look at TIMP3 in paraffin tissue sections. I see ab2169 is listed as being tested in IHC-P but there is nothing about the crossreactivity with TIMPS 1,2,4 versus ab11814 which is listed as not cross reacting with the other TIMPs but it doesn't state whether it works in paraffins. Any information about this that you could provide would be greatly appreciated.

ANSWER:

Thank you for your enquiry and for your patience. Regarding ab2169, it has not been tested for cross-reactivity with TIPM 1,2 or 3. However, we would be more than happy to provide you the peptide sequence for you to see if there is any homology with TIMP 1,2 or 3 if you are interested. For ab2169, the antibody has been reported to work in IHC but has not specifically been tested yet in paraffin embedded sections. The antibody's originator has suggested first trying frozen sections fixed with formalin before trying paraffin embedded tissue. If you have any more questions, please contact us again.

I am interested in your anti-TIMP3 antibody (ab2169). However, I need to know if this antibody crossreact with mouse TIMP3. Or since the antigen is a peptide of human TIMP3, can you release the information of this peptide sequence? Or how much homology between human and mouse sequence in this peptide region? Thanks.

Jun-Hsiang

ANSWER:

We do not know its crossreactivity with mouse. However, it does not cross react with rat, cow, pig and bird. On the other hand, it does crossreact with rabbit and baboon.