Dear researchers,
I need some advice from more experienced people, please. I have a bacterial strain which is producing both S-layer proteins and a bacteriocin with similar MW. How can I specifically extract S-layer (without the risk of having the bacteriocin extracted in the same time)? I cannot count on SDS-PAGE because of the similar MW.
Can someone help me?
Thank you!
Hi Roxana,
Maybe you can extract both proteins and perform a purification step at some point using a Resource Q or Resource S column. The Resource columns are high resolution columns that can separate your S-layer proteins and a bacteriocin based on charge...So if the isoelectric point (pI) of your S-layer is above 7, then try a resource S with a buffer having pH below 7 and likewise use a pH above 7 for a resource Q column to purify S-layer if the pI is below 7....
So you could use an affinity column first in which your S-layer protein/s have an affinity for followed by Resource Q/S purification.
Do not use the Resource Q/S column as a first choice - use affinity or so followed by Resource Q/S column for polishing prior to gel filtration.....
Hope this helps.
If both S-protein and bacteriocin are of same Mr, you can attempt their separation using a small [5 ml bed volume] DEAE-cellulose anion exchange column. This approach may solve your problem.
Secondly, run the extracted fraction on a Native PAGE to see whether you get one protein or more protein bands.
- Badges
- Science method