In PAGE, I prepared 4% stacking gel and 12% resolving gel. I am not getting sharp bands and its 'trailing'. What should I change to get sharp bands? Can you please help me?
What are you working on? Proteins or DNA? If proteins then try making 10% stacking gel as 15% resolving gel. Also, once the protein is stacked in the upper gel and is about to enter lower gel, increase the current. Good luck.
'Trailing' or streaking indicates too much NaCl in your samples. Reduce the salt concentration of your samples and this will go away.
For sharper bands, make sure the pH of your stacking and resolving gel buffers are accurate. Also, make sure the stacking gel is at least 1 cm.
Thank you Sujata and Christopher for suggestions. I will try the options and let you know again. Sujata : I am working on protein. Thank ypu very much.. !
Probably the concentration may low so either improve the conc of protein or go for silver staining
Hii Anup, Would have been much better if you mention what kind of proteins, from plant or animal origin. How much protein you are loading on PAGE? However, in general, band resolution could be improved by doubling the salt concentration in stacking and separating gels, but the gel must be run at lower voltages. During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best resolution. Good luck!
agree with Allah. It is better you show people the picture of gel. But no worry, it is usually a minor problem. For my samples (plant protein), I usually use 65C 10min instead of boioling for 5min (as mentioned by many protocols) for my samples in loading buffer before loaded them onto the gel. good luck as well.
Hello Allah, I m working on plant proteins specifically changes in proteins due to different stress. Thank you very much for your suggestions !!
- Badges
- Science topic
- Similar topics
- Biological Science
- Botany