For determining the protein molecular weight, normally SDS-PAGE was used but some researchers also use zymography. im confused with both things. please clarify the difference between these two methods.
Good answers so-far, just make sure you realise that in SDS-PAGE, the protein is not only denatured but also negatively charged, and that hydrophobic portions often accelerate the migration on the gel because they attract more SDS molecules. Zymography is harder, because you need to keep the protein native (so no boiling in SDS) and you need to adapt the conditions (the pH of the PAGE) depending on the IP of the protein, otherwise it might not enter the gel at all. On top of that, you need to know how to do the in-gel activity assay (for instance mixing a neutral protein with your PAGE so that it can be digested by a protease, or mixing starch with the PAGE so that it can be digested by an amylase, and than using stains to visualise the absence of the protein or starch). If it is a cytoplasmic protein, you may still need to use reducing agents to maintain enzymatic activity. If it is a secreted protein, you may want oxidizing conditions and avoid reducing agents. If it is a membrane protein, you may need detergents and it may be very difficult to handle on a non-denaturing gel. Usually zymography is only used to see if an enzymatic activity from a liquid-phase assay is caused by a single protein, or by a group of closely related proteins (or isoforms) that do the same job but run slightly different on a gel (due to a glycan modification, phosphorylation, or some small difference in charge).
In short,
1) SDS-PAGE works for the vast majority of the proteins, you don't need to know much about your protein, it denatures your protein to make linear strands coated with negative charges, and it gives you a rough estimate of the molecular weight.
2) Zymography tells you nothing about the molecular weight, and you need to know quite a lot about your protein before you can devise a strategy for non-denaturing PAGE, but it can tell you if the protein is enzymatically active.
:))
in addition to classic SDS-PAGE, zymography is good to detect the enzymatic activity of the protein of interest because you proceed with PAGE without protein denaturation!!
As rightly quoted by Fazio in zymography there is no protein denaturation. There is no use of SDS and reducing agents such as beta mercaptoethanol and DTT... As a result the separation occurs on the basis of charge and inorder to know the protein of interest you can incubate the PAGE in substrate to perform actiivity staining. This is normally done for enzymes. However in SDS PAGE the protein separation is based on molecular weight rather than charge. Hope I could answer ur query...
Zymography is usually done to know the activity of an enzyme by either running the sample on gel co-polymerized with substrate or by incubating the gel in buffer after the run is complete. After all this process, the gel is stained using suitable staining solution (amido-black in case of protease zymogram) and then the activity of the enzyme can be observed on the gel (a clear protease band appears against dark background of amido-black).
As rightly mentioned in the earlier answers that SDS or any other reducing agents are not added so that the activity of the enzyme can be observed in its native form. Opposite to it, SDS-PAGE gives the information about the molecular weight of protein(s).
Good answers so-far, just make sure you realise that in SDS-PAGE, the protein is not only denatured but also negatively charged, and that hydrophobic portions often accelerate the migration on the gel because they attract more SDS molecules. Zymography is harder, because you need to keep the protein native (so no boiling in SDS) and you need to adapt the conditions (the pH of the PAGE) depending on the IP of the protein, otherwise it might not enter the gel at all. On top of that, you need to know how to do the in-gel activity assay (for instance mixing a neutral protein with your PAGE so that it can be digested by a protease, or mixing starch with the PAGE so that it can be digested by an amylase, and than using stains to visualise the absence of the protein or starch). If it is a cytoplasmic protein, you may still need to use reducing agents to maintain enzymatic activity. If it is a secreted protein, you may want oxidizing conditions and avoid reducing agents. If it is a membrane protein, you may need detergents and it may be very difficult to handle on a non-denaturing gel. Usually zymography is only used to see if an enzymatic activity from a liquid-phase assay is caused by a single protein, or by a group of closely related proteins (or isoforms) that do the same job but run slightly different on a gel (due to a glycan modification, phosphorylation, or some small difference in charge).
In short,
1) SDS-PAGE works for the vast majority of the proteins, you don't need to know much about your protein, it denatures your protein to make linear strands coated with negative charges, and it gives you a rough estimate of the molecular weight.
2) Zymography tells you nothing about the molecular weight, and you need to know quite a lot about your protein before you can devise a strategy for non-denaturing PAGE, but it can tell you if the protein is enzymatically active.
:))
Hello Anandaraj,
The rationale behind using zymography is to see whether there are multiple froms of the enzyme called isozymes , present in the source organism.
The differences have already been explained above.
:)
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