question about an oddity - (Jul/14/2006 )
Hey, y'all! I just have a brain-teaser
I am purifying a recombinant protein...aren't we all? 
I have found that the eluted fractions come off the column fine, everything's happy, but I have seen a strange phenomenon when I go to run the gel. there is a precipitate that forms when I HEAT the samples
d'oh???
the fractions hang out in the fridge just fine. the ppt shows up in the crude lysate, the flow-through fraction, and the first wash fraction...and then in elution fractions 2 through 8, with more precipitate appearing where the concentration of recomb protein is highest. All I do is standard...25ul sample/15ul loading buffer, boil~8-10 minutes, load onto standard 12% SDS-page. the gels look a little smeary, but mostly they separate fine...
the real question is, why does the precipitate show up AFTER heating? prior to the boiling step, there is no evidence of ppt...this doesn't really make sense to me. has anyone else ever experienced this before?
I am purifying a recombinant protein...aren't we all?
I have found that the eluted fractions come off the column fine, everything's happy, but I have seen a strange phenomenon when I go to run the gel. there is a precipitate that forms when I HEAT the samples
d'oh???
the fractions hang out in the fridge just fine. the ppt shows up in the crude lysate, the flow-through fraction, and the first wash fraction...and then in elution fractions 2 through 8, with more precipitate appearing where the concentration of recomb protein is highest. All I do is standard...25ul sample/15ul loading buffer, boil~8-10 minutes, load onto standard 12% SDS-page. the gels look a little smeary, but mostly they separate fine...
the real question is, why does the precipitate show up AFTER heating? prior to the boiling step, there is no evidence of ppt...this doesn't really make sense to me. has anyone else ever experienced this before?
Hi Aimikins,
Heat as you would know heat would denature the protein.. so would turn all clumpy ppt. Exactly like what happens to an egg white or whole egg when heated.. clump. so that is what i guess is happening to yr protein too.
Do these fractions have high concentraion of protein.. cn u dilute them and load?
Do you really need to boil the samples to load on sds-page.. i mean logically yes without boiling sds may not coat the protein completely but whatever the sds can do without boiling should be fine for yr protein to run in the gel.. its works for mine (dont' ask me why.. i dunno;)) anyways, u can try that. or try boiling less.. if there is no other reason to boil.
And are u boiling in presence of DTT or beta mercaptoethanol? these would help solubilization i guess.
There can be several other solutions i guess.. but u can try these for now.. do let me know if they worked!
G'Luck!
Thank you for your suggestion!
actually, yes 2-ME is in the buffer.
the protein is at quite high concentration. I have decided that it doesn't matter...I did not use the gel to quantify, just to check purity of prep and make sure the rprotein was the right size, and I was able to see the appropriate band in all fractions so it didn't all precipitate.
the other thing that I guess might influence, is that the loading buffer added would change the pH of the solution, and this protein is very picky about what it takes to remain soluble...so perhaps it was the addition of the buffer and not even necessarily the boiling?
well, thanks again
A
actually, yes 2-ME is in the buffer.
the protein is at quite high concentration. I have decided that it doesn't matter...I did not use the gel to quantify, just to check purity of prep and make sure the rprotein was the right size, and I was able to see the appropriate band in all fractions so it didn't all precipitate.
the other thing that I guess might influence, is that the loading buffer added would change the pH of the solution, and this protein is very picky about what it takes to remain soluble...so perhaps it was the addition of the buffer and not even necessarily the boiling?
well, thanks again
A
Uh! "This protein is very picky about what it takes to remain soluble" - i wud use the exactly same words to describe the protein i m working with too.. he he!
Mine is a highly basic, hydrophobic one.. may be that makes it so selectively soluble.. what's up with yours?
G'luck!
The ionic interaction btw anionic SDS micelle and cationic protein can be contributing factor for ppt formation at right ratios. But I have a hard time to understand the temperature effect as described.
From Molecular Cloning Vol. 3 Third Edition Page A8.45:
"Preparation of Samples and loading the gel:
7. While the stacking gel is polymerizing, prepare the samples in the appropriate volume of 1x SDS-loading buffer and heat them to 100°C for 3 minutes to denature the proteins.
Extremely hydrophobic proteins, such as those containing multiple transmembrane domains, may precipitate or multimerize when boiled for 3 minutes at 100°C. To avoid these pitfalls, heat these samples for one hour at a lower temperature (45-55°C) to effect denaturation."
I quote and refer people to Maniatis all the time...I suppose if I read it more I'd have known that I use it as the bible for cloning purposes, I often forget it is also good for proteins
thanks, that's an awesome tip.
Cheeztoast, my protein is quite basic, hydrophobic also