4 replies to this topic

[Alsai]

Hi!
I try to make blots for MMP2 and MMP9  but I don't receive appropriate bands. In both cases I have bands on the same level: about 37 kDa ( like for GAPDH?) and no other bands. MMP2 should be at 72kDa and MMP9 92kDa.
I'm using Santa Cruz antibodies and for primary antibody dilution was 1:200, for secondary 1:500.
I'm using chemiluminescence for detection.
What went wrong?

Edited by Alsai, 17 December 2009 - 01:48 PM.

[bob1]

Any one of a number of things could be wrong... starting from "do your samples have MMPs in them?"  and finishing with "are you using the correct secondary?"

A bit of detail on your method would be helpful... Have you tried titrating the antibodies?  Does the antibody have a peptide to you can see if the bands are specific?  Have you done longer exposures to see if the correct bands came up?  What percent gel are you running?  Did it transfer correctly (ponceau and/or coomassie stain)? etc, etc...

[Alsai]

bob1, on Dec 22 2009, 01:00 AM, said:

A bit of detail on your method would be helpful... Have you tried titrating the antibodies?  Does the antibody have a peptide to you can see if the bands are specific?  Have you done longer exposures to see if the correct bands came up?  What percent gel are you running?  Did it transfer correctly (ponceau and/or coomassie stain)? etc, etc...

I'm running 10% gel, I checked transfer and it looks good. I use primery antibody in 1:200 concetration. I tried using secondary antibody in 1:500, 1:1000, 1:5000 and it doesn't make any diffrence. There are no other bands beside these on about 35kDa.

Primery antibodies are MMP-2 (C-19) sc-6838, MMP9(C-20) sc-6840.
Secondary antibody: donkey anti-goat IgG-HRP: sc-2304

[Gerard]

I've done done some work on MMP's years ago and what I remember that we had problems with indentifying the bands in our samples.
The problem was that the intensity of the bands didn't correspond with results we found in elisatests.
We did also zymograms and they showed different patterns.
I would advice to try zymogram to see if there are active MMP's in your samples and if there are matches with the bands in your blots.

[SCBT Technical Support]

Hello Alsai,

Greetings from Santa Cruz Technical Support.

It seems you have been trying to troubleshoot your Western Blots on your own, which is great. I assume that you verified that you have MMP present in your samples and that you are storing the antibodies properly at 4˚C.

I recommend that you contact us, either via phone (800-457-3801) or email (scbt@scbt.com) and we would be glad to help you troubleshoot your experiment to see if we have any advice to give. Since we want you  to succeed in your research, we are certainly willing to replace the products for you if we find that the antibodies are not performing according to our WB standards.

We hope to hear from you.

Technical Support

Santa Cruz Biotechnology, Inc.
800-457-3801 ext. 2
scbt@scbt.com
www.scbt.com