I am using A549 cells and trying to check the expression of MMP-2 and MMP-9. I got results for zymography for both the proteins. but not getting bands in western blotting. Antibody is working because others got bands for these proteins in their cells. I am not able to understand the problem. If anyone have idea about this problem please guide me.
Use the protein lysate for one lane as a positive control from other cells (in which others getting result) along with A549 cells lysate.
Hi Hina,
From your question, it is not clear what prep have you used for zymogram? Is it the cell lysate or the serum free culture media supplemented with ITS? MMP2 and MMP9 are both secretory and best results are found when you run the concentrated media (Usually 4-5X conc using now Millipopre, then Amicon YM10 centrifuge-based filters). This prep also is expected to give good WB signals for both proteins.
As suggested by Sangappa, you can definitely try other cell lysates known to express MMP2 and 9 in culture (May be PASMCs or RONA). Buying an MMP assay kit from Millipore could be a good idea as it comes with positive controls, antibodies and also the inhibitors, TIMPS. For MMP2 and 9. 10% SDS PAGE works well.
Unlike zymogram assay where heating/boiling and reduction by using DTT or beta ME to the samples is detrimental, for WB you have to reduce the sample and need to heat with LSB before loading into the SDS-PAGE.
Will wait for your feedback how it goes!
A 549 can produce MMP2 and MMP9, as well as some other MMPs or TIMPs, but the secretion of these different MMPs is rather low and above all very dependent on the state of cell A 549. This can result in a negative WB .
The zymogram, which is very sensitive, can reveal specific bands of the two MMPs while the WB is negative.
But be careful not to have serum trace in the protein extract from A 549, since these two MMPs are very abundant in the FCS used to culture A549.
And if the two MMPs, of bovine origin, are not revealed on the WB, it is because the antibodies anti MMPs, used to detect the human MMPs resulting from A549, do not cross with the MMPs of bovine origin.
I had the same trouble when I studied MMP12 twenty years ago.
hello Vicky - have you spoken to romany? she did WB for MMPs in her PhD and could help. I am not sure if she is still using her UoA email, but perhaps just email Jo and ask for her lab books?
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