I have seen a few papers mentioning like they have used urea, chaps, DTT and Pharmalyte for protein extraction. Is pharmalyte an ampholyte? What's the difference between a pharmalyte and an ampholyte? Can we use biolyte 3/10 ampholyte instead of pharmalyte for protein extraction to perform 2D gel electrophoresis? What are the other buffers that I can use to extract proteins for 2D gel electrophoresis?Can anyone please advice me on this?
Thanks in advance
You could use both pharmalyte or biolyte. Just be careful about the real concentration of these ampholytes. At low concentration, they help to create the IEF gradient. At high concentration, the might induce some problem of voltage during the focusing.
For a "good" 2DE, you need :
- enough urea (or a mix of urea and thiourea),
- a detergent such as CHAPS but you could try some others (cautious about the mix of various detergents because the effects can not automatically added)
- and DTT (or another reducing agent but not beta-mercatoethanol).
Thanks for the suggestion Stephane !!! It would be of great help to me if you have found some links or articles that have used biolyte for protein extraction for 2D electrophoresis,and if so please provide me with those.
Thank you very much,
pharmalyte or biolyte both are brand names for ampholyte. Both are same, but its always advised to chose the ampholyte from the same manufacturer of the IPG strips, as both the pH gradients of strip and ampholyte should match well.
Your choice of buffer is usually specific to your sample and usually ampholyte is not added during protein extraction, but mixed with processed sample just before adding to the strips.
Pharmalyte brand name of Pharmacia, Biolyte is brand name of Biorad and SERVALYT is brand name of SERVA, Germany. However, all are Ampholytes.
As Mr. Sourav explained we always make sure that we buy IPG strips and Ampholytes from same manufacturer. SERVA IEF reagents such as IPG strips and SERVALYT (ampholyte) are better compared to Biolyte (my personal observations).
for any IEF protocol two things are important: make sure protein lysate buffer contains enough protease inhibitors and remove excess salts before loading on the IPG strips
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