Hi Anju,

OK, I understand, but you should not try to separate according to size, you need another property, such as charge or hydrophobicity. IEF is fast and should do it if you just want to verify that you have two protein bands. If you want to isolate the two fractions, a small chromatography column will be most helpful.

Good luck!

For that we would need to know the exact masses (i.e. you'd use different gel for 43 and 44 kDa and another for 120 and 121 kDa).

Anyway, 1 kDa difference is too small to be separated by PAGE. Maybe isoelectric focusing could help.

As previously mentioned 1 Kda difference is too small to be clearly separated by SDS-PAGE. However we got same nice results increasing the % of acrylamide (up to 20%) or using gradient gels.

You might get some separation with a large sized gradient gel. Maybe.

Thanx Rosa.  Although  I have tried upto 15% but i ll try  again with 18 and 20 % if that can help me out.

Perhaps you could try in a native gel and could differentiate between them.

You can't. Not by PAGE anyways. If they're on separate lanes you could do western blot to the tag.

You could try 2d gel electrophoresis if they have different pl.

You may try tris-tricine buffer system for SDS-PAGE. It will sharpen your bands and thus make it easier to distinguish bands. However, it is more expencive, than tris-glycine buffer. An example of the protocol I used: http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.4.html

If you have access to MS instrumentation, I would suggest that you first verify the sizes of your two proteins. Electrophoresis is normally not a good approach for separating biomolecuels with such small differences. A simply ion exchange column will most likely be able to do this separation.

Hi Leif

actually my protein is a known protein of 57 kda . When protease cleaves it , last 11 amino acids are removed from it, so then theoretical mol. weight comes around 56 kda. That why I was thinking to differentiate it by electrophoresis.

Hi Anju,

OK, I understand, but you should not try to separate according to size, you need another property, such as charge or hydrophobicity. IEF is fast and should do it if you just want to verify that you have two protein bands. If you want to isolate the two fractions, a small chromatography column will be most helpful.

Good luck!

Increasing acrylamide % will not help. That will only shift the resolution to smaller sizes. That's really useless. Gradient won't help either. Gradient gel is great if you want to separate well both big and small proteins.

You will need something like 14% as long as possible and keep it running for as long as possible (ideally if you have pre-stained marker). But with 1 kDa difference I doubt you will be able to resolve them anyway.

http://www.protocol-online.org/biology-forums-2/posts/11794.html

What are the amino acids, that are cleaved off?

for this type of problem you have to go for 2d gel electrophoresis because sds-page is not that much sensitive for lower molecular weight difference.

U can try tricene Gel electrophoresis with 15-20% acrylamide

 You cannot use SDS-PAGE to separate these two proteins.  You should try Tricine SDS-PAGE (see the link)  or 2D gel electrophoresis if the isoelectric point is different between the two proteins. 

https://www.researchgate.net/file.PostFileLoader.html?id=52d99e47d3df3e89078b4593&assetKey=AS%3A272187611648000%401441905991523

anju bala i want to know you get your results ??

if yes then what method you used @

With SDS-PAGE, the best way is to use 7.5% polyacrilammide gels or at most 10%, run your gel as much as possible using a pre-stained protein marker to make sure that your proteins of interest do not come out from the gel.