Hi Omar, electrophoretic migration on PAGE is designed for globular proteins. Linear proteins, e.g fibrinogen, collagens, etc. run differently on gels compared to other proteins of similar MW.

Any proteins that are linear as supposed to globular will run weirdly. And yes, if your enzyme has a linear substrate attached to it, it will run weirdly.

My suggestion is to run an agarose gel (normally for DNA), but it is more forgiving if the sample being run is not globular.

Hope this helps.

Hi Omar

I don not agree with Steingrimur

SDS PAGE is designed to ONLY run linear proteins, regardless of their native state, because SDS plus mercaptoethanol and other components in the buffer will denature the protein to a linear form

You need to give more details about your methods in order to analyse your results in more detail, it coul be that the subtrate is bound, it could be that you just inducea a conformational change i the protein, but many things have to be considered before considering any possible explanation

Hi Leonardo, you are absolutely right, SDS-PAGE is designed to run denatured proteins made linear by SDS denaturation and reduction of S-S bonds by b-mercaptoethanol.

But I did not mention SDS-PAGE in my answer.

But that is not how Omar presented his question. He did not mention SDS-PAGE and since he is running an enzyme with a bound substrate, it should be obvious to everybody that he is not using SDS-PAGE.

Native PAGE is the way to analyze proteins bound to substrates and Native PAGE favors globular proteins. That is why I suggested agarose gels.

Hi Leonardo and Steingrimur

Fristly, thanks for your comments, and secondly, sorry since maybe I was not very specific. I am attaching an image where you can see what I mean. The gel is in SDS-PAGE format, so it does not contain any substrate. What I actually do is to incubate my protein (which is active on cellulose but actually is not an enzyme) with an insoluble form of cellulose isolated from celery vascular tissue and thereafter I run the supernatant of the reaction in the SDS-PAGE. What I see at the end is my protein of interest (the intense band) at a different MW.

In the attached image, the red line is where I normally see my protein, and the arrow is where I see it after incubation with the substrate. I have made the experiment lots of times....

Thanks in advance!

Hi Omar

I hate to see I was right. Anyways

What is the size of both your protein and substrate (estimate).

Also what are the concentrations?

In order to see a real shift in mass, you would need to have all your protein reacting with the substrate, which I am assuming is not the case. Normally your protein concentration would be so much higher than your substrate concentration. Is this right?

What I think is happening, is just that something in the substrate (not the substrate itself) is causing your protein to migrate differently in the gel (this is not to do with size, but maybe charge or denaturation degree).

Are you following all the steps for SDS page? such as adding same buffer to both conditions, boiling, fresh DTT, etc?

You could run a third control, protein plus buffer of the substrate, this may rule out any contribution from the buffer of your substrate

How did you express your protein? in E.coli ?  ist it normally glycosylated ? 

or  might your extract glycosylate your protein? 

glycosylation can interefere with the SDS-Uptake 

It is good practice to run your standards either side of your critical samples, that way any heterogeneity in the gel can be accounted for.

As Omar has mentioned knowing the recipe of your sample loading buffer and other buffers will help elucidate what is going on. It's probably worth running your cellulose extract on the gel too to see that you have a clean cellulose sample.

Do you incubate your control in the same way? i.e. is it a cellulose free control which under goes the same process?

You say your protein is not an enzyme but then that it shows activity, you need to be concise in your definition.  Are you trying to say that it's binding protein that displays other binding characteristics when it is bound to cellulose? Can you share the name of the functional family of the protein?

If the protein binds and retains a fragment of the carbohydrate the protein will have a hydrodynamic volume increase which will show as an increase in MW, this increase is significantly larger than the actual contribution in MW the carbohydrate.