Anti-TIMP3 antibody - Carboxyterminal end (ab39185)

LOT NUMBER Gr 8727-1 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM I'm obtaining bands above 30 kDa for ab39184 and ab39185 (TIMP antibodies). In the description of the antibodies ab39184 and ab39185 is: Anti-TIMP3 antibody - Carboxyterminal end (ab39185) WB: 1/1000 - 1/5000. Used under non reducing conditions. Detects a band of approximately 21 kDa (predicted molecular weight: 24 kDa). Anti-TIMP3 antibody - Loop 1 (ab39184) WB: 1/1000 - 1/5000. Dilution optimised using Chromogenic detection. Detects a band of approximately 24 , 30 kDa (predicted molecular weight: 24 kDa). My bands are above 30 KDa, what should I do? SAMPLE Type of sample: cell lysates of mouse PRIMARY ANTIBODY Primary antibody (If more than one was used, describe in “additional notes”): only TIMP3 (ab 39184) Concentration or dilution: 1:200 Diluent buffer: TTBS 1x (TBS 10x and distilled water) Incubation time: overnight Incubation temperature: 8ºC DETECTION METHOD ECL ANTIBODY STORAGE CONDITIONS as recommended on the datasheet. SAMPLE PREPARATION Lysis buffer: RIPA Boiling for ≥5 min? yes AMOUNT OF PROTEIN LOADED Protein loaded ug/lane or cells/lane: 30 ug/ protein ELECTROPHORESIS/GEL CONDITIONS Non reducing gel. Percentage of gel: gradient 5-20% TRANSFER AND BLOCKING CONDITIONS Type of membrane: nitrocellulose membrane Protein transfer verified: yes Blocking agent and concentration: milk 5% Blocking time: 1 hour Blocking temperature: room temperature SECONDARY ANTIBODY Secondary antibody: anti-rabbit IgG HRP conjugated ( Amersham NA934) Species: Reacts against: rabbit Concentration or dilution: 1:5000 Diluent buffer: TTBS 1x Incubation time: 1h Incubation temperature: room temperature Fluorochrome or enzyme conjugate: HRP Washing after primary and secondary antibodies: Buffer: TTBS Number of washes: primary, 3x (5 min.) secondary, 3x (15 min.) DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Concentration of antibody

ANSWER:

Thank you for taking the time to contact us.

I am sorry to hear you have had difficulty obtaining satisfactory results from these antibodies. The details you have kindly provided will enable us to investigate this case for you and also gives us vital information for our monitoring of product quality.

1) When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. For unknown reasons some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : Click here for the western blot image using ab9385 (or use the following: www.abcam.com/ab9385).  

2) I am unsure if the bands you are observing are around 80 kDa, as the resolution of the higher molecular weight marker bands is suboptimal. In case no bands have run out of the gel and you count from the bottom, the TIMP3 bands could also be around 60 kDa. If that is the case they may represent multimers of the protein (32 kDa dimers were reported according to the literature) or complexes with metalloproteinases. Therefore, I would recommend boiling the samples for more than 5 minutes (e.g. 10min) which may help to reduce disulfide bridges and break up complex structures.

We are happy to offer this technical support. In the event that the product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.  

Hello,

Thanks for your reply.

Attached is a file of my blot that was probed with the anti-TIMP3, ab39185.

In this blot we included some conditioned medium from melanoma cells that we know have TIMP3 protein (proteomic data).

We did probe this blot with anti-TIMP 1 purchased from other company and the band was at the correct molecular weight.

So we know the OVC extracts are OK.

Your suggestion that the 50kDa band may be a TIMP3 dimer, is acceptable but I have not came across any reference of its existence.

If you know of any references, please pass along :).

Would it be possible for you to send a small aliquot of other anti-TIMP3 ab that has been used by other researchers in Western blotting for us to confirm if indeed the 50kDa band is a TIMP3 dimer.

ANSWER:

Thank you for providing the additional information and western blot data.

It appears that ab39185 detects a protein of the expected molecular weight in the conditioned medium however the 50kDa band is seen in the OVC tissue extracts. And the band in the OVC samples seems to be quite specific.

I have found TIMP3 can form dimers but, as I've found, only regarding the following:

"Mutations to TIMP-3, all of which introduce an extra cysteine residue into exon 5 (which forms part of the COOH-terminal domain of the molecule), have been linked to the macular degenerative disease Sorsby’s fundus dystrophy"

in: The Journal of Biological Chemistry, 273, 16778-16781.

Also we do not have free samples available.

Hi,

I recently purchased anti-TIMP3 antibody (AB39185) from your company for Western blot.

I am detecting a very strong band at 50 KDa in ovarian cancer tissue samples. A very weak band of the predicted size for this ab, 20 kDa could be seen only with a long exposure. What can you suggest what could be the reason of this strong

band?

ANSWER:

Thank you for your enquiry.

Just a few additional questions regarding your western blot:

1. Have you used any other antibodies for western blotting with this ovarian cancer tissue extract? If so, what were the results?

2. The 50kDa band you see may be a TIMP3 dimer. Have you proped any other human or mouse tissue or cell lysates with ab39185 as a comparison?

3. It might be helpful to view your results. If you could, please forward along an image of your western blot.

Thanks very much for the additional information.