Hi there,

What is the actual MW of the protein?

Dear Ninna Koho ,

The higher MW in your zymograph could be due to several factors involved:

-You could have a multimer of your protein

-A dimer of your protein associated with some sort of cofactor

-A dimer of your protein

-A monomer with some sort of cofactor

These are possible reasons why the MW might be higher. See for a nice example with explanation:

http://protocol-place.com/assays/gelatin-zymography/gelatin-zymography-band-interpretation/

The whole purpose of using zymography is to keep or renature your protein in a native (active) state. On the contrary to ‘classical’ SDS page based methods like your western blot where proteins are denatured and because of the addition of the disulphide bridge breaking agent it breaks possible dimers of your protein. Because of this the MW might be lower, in order to test this you could run without the addition of mercaptoethanol?

See for an example where Western immunoblotting detected only the monomeric form (of in this case the protein MMP-9), while under non-reducing conditions, zymography detected the dimeric form:

Lam, C., Jamerson, M., Cabral, G., Carlesso, A. M., & Marciano-Cabral, F. (2017). Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system. Microbiology, 163(10), 1436-1444.

https://www.microbiologyresearch.org/docserver/fulltext/micro/163/10/1436_micro000537.pdf?expires=1570446363&id=id&accname=guest&checksum=4BFD47F98FCB5D4B792CE610EDD837D0

Anyway I hope this helps you a bit further.

Best regards.

Thank you all!

Agree with Rob above. If you're worried that the 2 signals may correspond to different proteins, try to minimize differences by not including reducing agents during your SDS-PAGE. Regardless, since both gels are likely quite different, don't expect proteins to behave the same in both (e.g. are your markers behaving the same? Probably not). An option you may have is to perform WB on your zymograph (i.e. run your zymograph followed by tansfer and immunoblotting).

your zymography should be done in native PAGE to maintain the native, active, structure of your protein. why do you have SDS loading dye for a zymography experiment? It should denature your protein (depending on concentration). SDS and a reducing agent (mercaptoethanol or DTT) should be absent. You cannot estimate the molecular weight from a native PAGE (it will run as an intact quaternary structure). How do you estimate its size in zymography?

A reducing agent used for SDS-PAGE is added to break your disulphide bonds so, again, you can estimate the molecular weight. Leaving the S-S bonds intact (no reducing agent) will make your protein run differently, usually slower, because the polypeptide chain is not linear.

If you are running odd mixtures of native and SDS and reducing conditions you will get odd results.

Immunoblots should be done in SDS-PAGE of course, but there are exceptions if you need to identify a particular band in a native gel for example.

First, as mentioned by others, you want to compare your zymography bands with the bands on SDS-PAGE/Western blot where the gel is run in the absence of reducing agent. Then, if you do that, there is still a possibility that the migration of your enzyme of interest will be different. This is well-documented by others who incorporate protein substrates of proteolytic enzymes into their zymography gels, presumably because the enzyme-substrate interaction results in slower migration. To get around that, we devised a transfer zymography method to determine the size of the enzyme of interest. In essence, the method consists of running your sample with enzyme on non-reducing SDS-PAGE without substrate in the gel. Another gel of the same size, with substrate incorporated into the gel should be prepared the day before and kept cold. Then, using the same apparatus that you would use for protein blotting, press the two gels together and do an electro-transfer from the gel with the enzyme (having traveled without interference on the non-reducing SDS-PAGE) to the gel that has the substrate (that has not been subjected to any electrophoresis). After the transfer, the reaction is allowed to take place by incubation in buffer and temperature conducive to reaction, then visualized. In our case, we study proteolytic enzymes, so staining with Coomassie will show the clear bands where the transferred enzyme has digested the substrate. The molecular weight for your enzyme of interest that you determine in this manner will be the same as the molecular weight for your enzyme of interest on your Western blot, although the clear band will not be as sharp. You can get details in the following: Pan et al (2011) Anal Biochem 411: 277-283 and a detailed protocol in Pan et al (2017) chapter in Zymography Methods and Protocols, Methods in Molecular Biology 1626: 253-269.

Hi Ninna Koho ,

Our protein has several sulfur bridges

What else could be the reason for size differences in zymo and western?

Using B-mercaptaethanol you will disrupt the dissulfidic bridges and allow your protein run freely in you gel. The overall effect of SDS and BM is linearize the protein, allowing a faster run between the gel pores. Your result is expected!

Best,

Jacó