[WTK14]

Hey all,

I am working on a project involving an extraction of an allergen protein. I was able to extract it from the raw source, and it is currently in a freezer. My work requires me to analyze it via several methods of analysis. I am having trouble running my protein via UV-Vis; the main problem being that my protein just doesn't seem to be soluble in any solvents I use. So far I've tried acetone and methanol (which was used in the extraction process), but i've had no luck with sonication and stirring. Does anyone have any potential suggestions?

THanks

[Arkcon]

The protein may have become denatured, and may no longer be soluble.  What do you know about this particular protein?  What is its natural state -- I mean to say, where is it found?  Things like pH, ionic strength can't be ignored when a protein is extracted.

[Yggdrasil]

Using acetone and methanol for protein extraction seems like a fairly harsh method that would denature the protein.  Are you examining peptides or full proteins?

Sometimes denatured proteins can be resolublized in a solution of 7M guanidine-HCl, so that could be something to try.

[rwiew]

Yes, why would you try to dissolve a protein in organic solvents? That makes me feel sorry for that protein, cruel.

Aqueous buffers? What is this measurement method? Nanodrop?

[WTK14]

Sorry, I am inexperienced in biochemical techniques. I am currently examining the allergen phl p 5 from timothy grass, and have been having a hard time gathering information (due to a lack of access to papers). If I were to go try an acid, I am worried about the lack of recovery of the protein after dissolving it in solution. Unfortunately, I have to retrieve the protein post analysis and using a straight up acid would be problematic on my rotovap. I was thinking about doing a mixture of 10% TCA in acetone. Any thoughts?

[Arkcon]

A quick Google tells me that many people get their phl p1 and p5 allergens from recombinant sources, and purify them by ion exchange chromatography.  If you can find a vendor, they may describe storage conditions, and you can use that as a guide for how to handle these proteins.  A buffer at a certain pH, and a certain ionic strength is probably necessary to prevent the protein from denaturing.  Although you really should try to read some literature on these proteins if you really want to work with them.

[Babcock_Hall]

My understanding is that trichloroacetic acid is often used when one wants to precipitate a protein:  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771300/  I think you should be staying away from organic solvents and TCA if you want your protein to remain in solution.

[Yggdrasil]

Sorry, I am inexperienced in biochemical techniques. I am currently examining the allergen phl p 5 from timothy grass, and have been having a hard time gathering information (due to a lack of access to papers). If I were to go try an acid, I am worried about the lack of recovery of the protein after dissolving it in solution. Unfortunately, I have to retrieve the protein post analysis and using a straight up acid would be problematic on my rotovap. I was thinking about doing a mixture of 10% TCA in acetone. Any thoughts?

You cannot treat protein samples as you would most chemical substances you encounter in a chemistry lab.  Proteins are extremely sensitive to the conditions under which you place them.  Proteins almost always should be in aqueous solution, and small amounts of organic solvent will often denature them and destroy their activity.   In addition, not any aqueous solution will work, most proteins require a fairly narrow set of temperature, pH, salt concentrations in order to maintain their activity.

Thus, trying to rotovap your sample is a bad idea for a number of reasons.  First, you don't want your protein to be in any of the typical solvents removed by a rotovap (e.g. acetone).  Second, heating proteins is generally a bad idea.  If you want to recover your protein in solid form, lyophilization is a much better approach.

Here is a document with some tips for handling protein samples.  It's not entirely comprehensive, but it has some good pointers if you have little biochemistry experience:
http://stanxterm.aecom.yu.edu/wiki/index.php?page=Protein_handling

In addition, consulting the published literature to see under what conditions others store and use the protein is a must.  Remember the old addage: a day in the library can save you a week in the lab.