MMP9 protein (Human) (ab39308)

I did an MMP9 western blot using different amount of total protein/well (20ug-50ug, rat ovarian lysates) along with a 30 ng of MMP9 protein from abcam (cat# ab39308) as positive control. The antibody was cat#ab58803. Samples were reduced with DTT, heated for 10 minutes at 70oC, then loaded on a 7.5% polyacrylamide gel.

The MMP9 protein (ab39308) yielded one band between 82 and 122 Kda (the expected size is 92 Kda, so I think that’s probably the right size).

The sample yielded 2 bands, both below 82 Kda.

I attached an image of the results.

Am’I detecting MMP9 in my sample?

If so why are the bands sizes lower than expected (expected 92 Kda)?

The antibody data sheet in the application section states that the detected band is approximately 92 Kda, but the predicted is 78 kda. What does this mean?

If I have to consider the predicted size (78 kda), why the abcam MMP9 protein was not that low?

ANSWER:

MMP-9 has a propeptide domain that is cleaved just after the "Cysteine switch" region, releasing the first 100 residues.
The full length protein is 707 amino acids, with a predicted mass of 78.4 kDa, but glycosylation makes the 78 kDa protein
run at about 92 kDa on reduced SDS PAGE gels, and about 95 kDa on nonreduced substrate gels (gelatin zymograms). The
cleaved mature protein is 607 residues, with a predicted mass of 67.2 kDa. If this form is glycosilated then the protein runs around 85 kDa, and in a substrate gel around 88 kDa.
I hope this helps to explain your western blot results. Please feel free to contact me again if you have any further questions.

need to perform gelatin gel
zymography to see MMP9 activity for which I need a MMP9 standard. Your
website does provide information about the MMP9 human and mouse
protein. However, I wanted to know if it is the active enzyme that you
provide, as I do not need to quantify the protein, but need to check
for activity. Kindly do let me know at the earliest.

ANSWER:

Thank you for contacting Abcam.

The ab39308 protein is largely in the zymogen form. The enzyme does not need to be activated before running on an SDS PAGE substrate gel (gelatin zymogram), as the SDS acts to activate some of the MMP-9.
Please let me know if there is anything else I can help you with.

Please update me if you have answers to my questions.

ANSWER:

Thanks for your email.
I apologize for the delay. I am currently awaiting a reply from the lab that supplies us ab58803. I have contacted them again just now and encouraged them to reply as soon as possible.
Thanks for your patience. I will forward you any information as soon as I receive it.

Dear technical team,

Our customer needs MMP9 protein active and inactive form.

I have searched MMP9 but couldn't get information about active and inactive form.

Let me know, please.


Best regards,
Youngjin


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ANSWER:

Thank you once again for your enquiries regarding our MMP9 proteins.

To follow up, I now have further information regarding the following proteins:

ab39308 MMP9 protein (Human)
ab39309 MMP9 protein (Mouse)
ab41067 MMP9 peptide (Carboxyterminal end)
ab41068 MMP9 peptide (Aminoterminal end)
ab41069 MMP9 peptide (Hinge Region)
ab41070 MMP9 peptide (Propeptide domain)

I can confirm that these peptides are not active, and are short immunogenic sequences.

I can also confirm that the following are not tested asbiologically active, as discussed in my previous email which I hope you have recieved.

ab82938 MMP9 protein (His tag)
ab82938 MMP9 protein (His tag)
ab82955 MMP9 protein (His tag)
ab82957 MMP9 protein (Ready to use SDS-Page sample)

If you have any further questions, please do not hesitate to contact us.

I am looking for recombinant proteins for MMP-2 and MMP-9 for gelatinzymography.

Do you have any and if yes how much are they?

ANSWER:

Thank you for contacting us.

We do have several MMP proteins in our catalog.

We have for MMP2:

ab39302 (human):Full length Human MMP2 protein (purified from human fibroblast cell culture media). It has not yet been tested in zymopgraphy. (10 ug for $274.00)
http://www.abcam.com/MMP2-protein-Human-ab39302.html

ab39304 (rat): purified from rat lung epithelial cell culture media. The enzyme is supplied as a mixture of the zymogen and processed enzyme. This protein was purified from rat lung epithelials, and is free of its endogenous inhibitor, TIMP2, and other matrix metalloproteinases.
It has been tested in zymopraphy. (10 ug for $274.00)
http://www.abcam.com/MMP2-protein-Rat-ab39304.html

We have for MMP9, but none of these proteins were tested in zymography:

ab39308 (human): Purified from cell culture media of stimulated human fibroblasts, this protein is free of its endogenous inhibitor, TIMP1, and other matrix metalloproteinases. (10 ug for $274.00)
http://www.abcam.com/MMP9-protein-Human-ab39308.html

ab39309 (mouse): Metalloproteinase purified from cell culture media of BHK cells transfected with Murine 105 kD Gelatinase (murine homologue of Human MMP9). Purified from cell culture media of BHK cells, free of its endogenous inhibitor, TIMP1, and other matrix metalloproteinases.
(10 ug for $274.00)
http://www.abcam.com/MMP9-protein-Mouse-ab39309.html

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.