After running E. coli transfection protein (molecular weight 15 kDa) on SDS-Page and performing Coomassie brilliant blue staining, using 12% separating gel and 5% concentrating gel, voltage 100V, current 35mA, staining with 50ml staining solution A+B for 2 hours, and decolorizing overnight with MQ. Why can only bands with molecular weights above 20 kDa be seen, while the band at 15 kDa is blurred and unclear? How to solve it?
Hello Liang, this might be happening due to the low intensity as a result of loading low sample volume onto the gel-well or the staining solution is overused or it is not binding to the proteins, you have also used only water for destaining which might not work sometimes for clearing the background stains properly. To overcome this you can use a colloidal CBB (G-250) solution (0.1% (w/v) CBB G 250, 5% (w/v) aluminium sulphate, 10% (v/v) ethanol, 2.35% (v/v) phosphoric acid) staining solution, I have observed good results using this one. Also, for destaining try 50% methanol, 10% acetic acid in water. Hope this helps!
Your 15 kDa band is unclear because:
Low protein amount: Load more protein or concentrate the sample.
Staining issues: Use a more sensitive stain like silver staining.
Diffusion: Fix the gel before staining and reduce destaining time.
Gel problem: Verify gel preparation or use a gradient gel.
Degradation: Use protease inhibitors during sample prep.
these steps can help resolve the issue......
To improve the clarity of bands, increase the amount of protein loaded onto the gel . Using a 15% gel can also enhance separation, especially for lower molecular weight proteins like 15 kDa. Avoid waiting for higher molecular weight bands to resolve and focus on optimizing the resolution of lower bands.
This is most probably due to a degradation of the protein. Check your sample condition. Another reason might be the less sample concentration. Try with higher concentration of sample. Or else, if the high MW protein bands are not required, take 15% resolving gel and run at 25 mA current. Sometimes low concentration proteins get diffused during the run in 12% gel.
Dear Liang Tang
as Junaid Nazir suggest, for a so small protein is preferable the use of an high % of acrilamide as 15% or 17%.
good luck
Manuele
Try it with 15 percentage gel. And first make sure that the marker of near 15 KDa should be visible.
Hello Liang, are you sure your protein is being expressed? More context would be useful for troubleshooting. Could you kindly share your protocol and what the goal is? Thanks!
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