I ran an SDS-PAGE at 150 V for 55 minutes in an attempt to concentrate pure samples of my protein (65kDa).
I observed a thick band at the expected size. However, I also observed intense bands of lower molecular weight than my expected protein. These bands did not appear during purification.
Is this typical of an SDS-PAGE after concentrating pure protein? What can I attribute this to? Does anyone have any tips for troubleshooting?
Hi,
It sounds like some kind of degradation occurred during sample preparation and/or analysis.
Is your protein of interest a monomer? How did you concentrate your protein sample? And what do you mean by 'These bands did not appear during purification': how did you purify your protein, also using a size-based method?
Hope this helps.
Hi there,
It seems your purified protein is proned to degradation. A picture of the PAGE should help. If you have discrete intense bands its worthwhile trying to identify the peptides to figure out what kind of digest actually occurs.
Hello! Thank you for your input.
My protein is a monomer and purification was performed using a HisTrap column. SDS-PAGE of fraction elutions didn't show the other bands.
Can we assume that the protein that you are purifying does not have proteolytic activity? Did the solution contain reagents to inhibit proteolytic digestion? What were the sizes of the other bands? Compared to the band of expected size, how intense was the staining of these other bands? If the bands were discrete (not a smear) but not so intense, it could be that you did not see them when you did SDS-PAGE of the fractions, but ended up concentrating them just as you are concentrating your protein of interest.
I agree with Anna Tan-Wilson.
If you know the total protein concentration in one of your fractions you could try to load the same amount of your concentrated protein on an SDS-PAGE. This will likely show that your protein is "pure".
As an example:
You load 2 ug and 5 ug of your protein and this could look 99% pure on a SDS-PAGE. When overloading the gel with 50-100 ug you might start to see other bands and realize that your protein is only 80 % pure.
Kind regards,
Simon
If you have an antibody against your protein you could use western blotting to distinguish between proteolytic digestion and other impurities.
If degradation is ruled out, then it is possible that you are concentrating the contaminants (as Anna pointed out). You can check by silver staining as these may not be visible by Coomassie staining (assuming you have used Coomassie). I have purified several His-tag proteins, and they are often not pure with just one step of purification by the His Trap.
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