I have extracted surface layer protein from lactobacillus with 5M LiCl, dialysis against distilled water for overnight (approximately 20 hours) (5-10C), and freeze dry them. However, I found that some of the freeze dried powder will turn into liquid again (yellowish brown colour) and they cannot freeze into solid at -18C, thus I cannot freeze dry them again. Are they remains of MRS broth and how do I freeze dry all my proteins? I have tried to store the freeze dried proteins at -20C and room temperature (25C) and this problem still exist.
You did not provide much information. Are your extractions from cell pellets? You are presuming the product extracted is protein; have you confirmed that? (what about carbohydrates?). As far as remnant of MRS broth....it contains Tween 80 (non-ionic surfactant) which won't freeze....could that have been extracted? Are you saying your freeze dried material changes back to liquid form on it's own? Sounds like a very hydroscopic substance absorbing moisture from the air. I don't think Tween 80 can be dried down....it is water soluble, and if extracted, I would presume it would condense down to a thick liquid.
The presence of their lipid anchors and/or the Tween 80 might be expected to make your samples behave this way. Did you do the multiday dialysis against water these preps usually require? Tween 80 has a cmc in water of 12uM and is thus very hard to remove by dialysis.
Thanks Dr Peter M Muriana and Dr Edward Michelini for your reply. Yes the extractions are from cell pellets and i had run bradford assay and SDS PAGE to confirm the presence of proteins. I also believe that other substances are also been extracted by this method. I think your kind suggestion is true that part of the Tween 80 is not dried by the freeze drying process. I'll try it again with multi-day dialysis as suggested by Dr Edward Michelini. Is there any other method that i can try to remove Tween 80?
Thank you for the suggestion, it helps me a lot.
Have you tried the general precipitation methods such as methanol/chloroform, acetone, etc to purify proteins?
We use the first precipitation method to remove SDS in protein sample before LC-MS/MS.
The links give more information about methods for protein purification :
http://onlinelibrary.wiley.com/doi/10.1002/elps.201000197/pdf
http://www.sciencedirect.com/science/article/pii/S0167701215000056
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