Greetings..
I am using Akta Pure system for my protein purification. I have optimized buffer system for protein elution. However, this time I got broad peak while eluting sample. For confirmation, eluted fractions were subjected to SDS-PAGE and desired protein bands were distinct on gel. However, the concentration of protein is 0.871mg/mL, which is less than expected. 5g of cells pellet was used for protein purification, which in last resulted in just 0.871 mg/mL concentration. Due to less concentration, I couldn't proceed to SEC. Is there any link between peak size or shape and eluted sample concentration ? Any suggestion in this regard will be highly appreciated. Thanks
The reason you got a broad peak is due to the lower concentration of your protein. Higher protein concentrations produce sharp peaks. Were there any undesired protein bands in SDS-PAGE?
There should not be a problem with broad peaks as long as they are pure. If the broad aspect of peaks is the only bothering issue, you can pool the fractions of interest and concentrate them using ultracentrifugation making sure the Cut Off MW is much less than your protein's size. For SEC, low concentrations are not usually a problem; we routinely inject amounts as low as 20ug of proteins, but this depends on the column. You can also simply increase the sensitivity of SEC by monitoring the elution at a wavelength of 215 nm (along 280nm), this way; you should be able to detect lower amounts of proteins. Broad peaks could be due to low ionic strength of the elution buffer, low slope of the elution gradient, non-optimal pH, too long column or tubing system (with large inner diameter). You can get broad peaks with old, poorly packed or defective columns, but I assume you do not have this problem for other proteins. Regarding the low yield, I am afraid I do not have much experience in that area.
Mehwish Iftikhar Getting a broader peak while sample elution may be due to the reason that your protein has different oligomerization states irrespective of the concentration of your protein. Dynamic Light Scattering technique can be adopted to find the dispersity of your protein sample . My suggestion would be to play with different buffer conditions which may bring your protein in more stable environment with less polydispersity.
Thank you everyone for your valuable suggestions.
1. Yes there are undesired protein bands but they seems to be very light Tushar Chakraborty Tushar Chakraborty
2. Dear Shoaib, I have concern about ultracentrifugation timings as it can effect the protein stability. Can you please tell me how much time (maximum time) we need to concentrate protein. I have performed three purifications by varying buffer systems and I agree, that might be my elution buffer isn't optimize or have some problem. Can you kindly recommend any literature or protocol for it Sohaib Mahri
3. Dear Ruby, my protein can not form dimer instead its twin protein has the ability to form a dimer or tetramer. Can you please recommend specific article using Dynamic Light Scattering technique for proteins. Ruby Sharma
The broad peak indicates the impure protein ...So the acute and sharp peak result from pure protein which doesnot any other materials or impurities with it..
Mehwish Iftikhar Most proteins we worked with are quite stable and are not affected much by the moderate stress caused by ultracentrifugation; the only way to know your protein is by doing it. The time depends on the concentration you want and the ultracentrifugation units you use. You can start with the recommended speed for 10 or 15 min and see how much volume is left then adjust based on that. We use Vivaspin® units; the recommended speed is around 4000 x g (follow the guide for your specific unit), MWCO must be less than half the size of your protein (e.g., < 10 kDa for proteins of 20-30kDa). Still, in our experience, we lose a significant amount of protein, so we go lower (e.g., MWCO 3-5kDa for proteins of 20-30kDa), the lower the MWCO, the less the loss but, the longer time you need for concentration.
For elution, you can find good suggestions on this free book “Strategies for Protein Purification Handbook” google it.
Thank you so much Sohaib Mahri for detailed answer. It really helped me out and I am now considering all aspects.
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