I am running a 12% SDS-PAGE gel with whole cell lysates. The problem is with lower molecular weight proteins (lower 1/4th of the gel) which lost their visibility after destaining. What could be the probable reason?
SDS can interfere with anionic dyes, like Coomassie Blue. If that's the case you can solve it by increasing the concentration of the dye.
With Coomassie stains, always try to leave a pale background. That way you, and everyone else seeing the data, know that the gels were not over destained. As CBB is reversible, just add a couple of drops of the stain solution to the destain to get a pale blue collar, and then leave gently shaking overnight.
Thank you. I will try both of your suggestions. One quick question does size of protein also plays role in staining?
The size of the protein shouldn't matter when it comes to staining, but if there is not enough dye or the SDS is interfering, it probably affects low molecular weight proteins more since they travel a longer path through the gel.
HI
As far as the SDS PAGE separation, gradient gels are the way to go. I often separate a 15 kDa protein on the 4-20% gels. If indeed you mean Western blot, and you miss the low molecular weight protein after transfer, try placing two membranes rather than one and see if your markers had passed to the membrane further away from the gel, suggesting the transfer is too long. Also, stain your gel after transfer and see if the entire marker has left the gel.
Low molecular weight proteins not adequately fixed after SDS-PAGE.; Use 20% TCA or glutaraldehyde as fixative instead of 40% alcohol and 10% acetic acid
hope this help
You could also try for silver staining if the low molecular weight proteins are expressed in low amounts.
On a12% gel, small proteins are well embedded. With AgNO3, some proteins having their own affinity. Perhaps, the destaining is too strong.
First, what sort of dye you used? Coomassie Blue, AgNO3, others?
- Badges
- Science method