Hello Researchers,
I am working with yeast whole proteome but after a lot try I only get spots in region between pH 4-6 using pH 3-10, 7cm strip, following BIO-RAD standard protocol. What could be the probable reason? How could I get get more spots on other pH as well?
- Maybe there is a problem with the carrier ampholytes in your sample. Is the concentration of the ampholytes correct? If possible, use a new batch.
- What protocol do you use for protein isolation from yeast cells?
- Could you attach a picture of the 2D gel? It would make the diagnosis of your IEF problem easier.
Regards, Christian
Salt content can be a problem... but if you could attach a picture, it is more likely that i can say more..
/Joakim
Thanks for replying.
Here, I have attached the image of gel. Hope it will work.
I am following CSHP, 2011 "Preparation of extracts from yeast" by Richard J. Simpson. Following 2 D cleanup from GE Healthcare.
This problem is interesting. Have you checked the theoretical pI of your protein? Sometimes they are even in this range. Furthermore, the less abundant proteins could be at the strip end and you could not detect them. Have you well leveling the strips on polyacrylamide gel? If the edges are not well leveled, the proteins in this region can not enter the gel.
The strip size could be another problem. The 7 cm strips not allow a good resolution. You could also increase the focusing time. If it is not enough, the proteins could not migrate to the strip edge.
Hi,
Despite the low quality of the attached picture, i could guess the problem of your protein migration, since you find a vertical or horizontal lines, you should check your focusing time although your two equilibration steps
here is a homepage that you might find interesting:
http://www.proteinsandproteomics.org/content/free/protocols_1/pro03.html
You could think about improving the staining procedure. A silver stain could reveal whether you actually have more protein spots in your gel (your gel was not stained by using silver nitrate, I suppose?).
Good luck!
Thanks all for your reply. I will definitely try all your suggestions.
@ Christian- Yes, it is commasive stained not silver nitrate. I will also try that. Thank you.
Hi Apurva,
You have got vertical streaking in your gel which suggests problem in 2nd dimension. Try leveling the strip properly on polymerized gel. Also you can increase the amount of protein loaded on the strip a lil bit to see if number of spots are increasing. Bring sealing solution to normal temperature at the end and then pour on strip. Too hot sealing solution also effects number of spots. You can also use colloidal commasie for staining as it is more sensitive.
All the best!
Hi Apurva..
The possible reason may be that the proteins in the other range are low in abundance or of low molecular weight which could not be visualized on 2-D gel. Try taking more amount of protein and increase the percentage of SDS-PAGE gel a bit.. Other reason is staining.. if it is coomassie blue, it is not that sensitive.. try with others like silver staining.. hope it would be useful..
Hi
I was working on some bacterial samples and facing the same problem. I have used narrow pH range strips for the better resolution. The possible reason may be that majority of proteins are in that range only and low abundant proteins could not appeared because of low sensitivity of staining. Try to use silver stain instead of coomassie staining.
Hello Apurva
I worked with plant protein samples and I too had faced these problems. You can use narrow pH range strips but before that try to ensure that while performing the second dimension there is no gap between the pH strip and the PAGE gel. Several times it happens if there is gap protein get collected at some places and run accordingly, so the interface should be carefully taken care.
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