samples floating out of SDS-page gel well immediately after loading - (Jun/21/2007 )
Hello all-
I am doing protein purification of Wnt3a. I ran my protein sample on a superdex 200 prepgrage 10/30 column and collected elutions in 1X PBS, 1% CHAPS (a detergent). I wanted to run all of my elutions on a gel and coomassie stain it to figure out which elutions have protein. I took some of my elutions and added 5X laemmeli sample buffer to the proper 1X concentration. and i started loading them on a 10% acrylamide gel and i was able to load elutions 1-14 fine. Then i started to try to load elutions 15-26 and they would just float out of the well. I made sure I had added 1X SDS running buffer to the chamber and got a new gel. Also tried using a 5X sample buffer that someone else had made to make sure that the sample buffer wasn't the problem and again the samples would just float out of the well. No one in the lab can explain why this is happening. We think that there is something in my elutions that is causing the problem but aren't sure what it could be. It should also be noted that I ran another aliquot of this sample run through the same type of column but in a different lab on a different purification machine and was able to run it on a gel and coomassie stain it.
Hi,
Well, I have to be honest and say that this is weird. I have only had one thing fly out
of a well - genomic DNA. I assume this is not the case here.
Try adding an equal volume of 100 % glycerol to
your sample, this should keep it in the well unless there is organic solvent (eg. ethanol)
contamination of your sample. If an equal volume of glycerol doesn't keep
it in the well your sample is most likely contaminated or contains long strands of genomic DNA.
I am doing protein purification of Wnt3a. I ran my protein sample on a superdex 200 prepgrage 10/30 column and collected elutions in 1X PBS, 1% CHAPS (a detergent). I wanted to run all of my elutions on a gel and coomassie stain it to figure out which elutions have protein. I took some of my elutions and added 5X laemmeli sample buffer to the proper 1X concentration. and i started loading them on a 10% acrylamide gel and i was able to load elutions 1-14 fine. Then i started to try to load elutions 15-26 and they would just float out of the well. I made sure I had added 1X SDS running buffer to the chamber and got a new gel. Also tried using a 5X sample buffer that someone else had made to make sure that the sample buffer wasn't the problem and again the samples would just float out of the well. No one in the lab can explain why this is happening. We think that there is something in my elutions that is causing the problem but aren't sure what it could be. It should also be noted that I ran another aliquot of this sample run through the same type of column but in a different lab on a different purification machine and was able to run it on a gel and coomassie stain it.
There def should not be any DNA in there because the Wnt3a protein is purified from conditioned media(its a secreted protein) and not from lysed bacterial or other type of cells. maybe the problem is some sort of contamination of ethanol or other such thing from the column? I am going to try a buffer with more glycerol tomorrow to see if this solves the problem but just wanted to see if anyone new what the issue could be and how it could be resolved. Other wise looks like I have to start over from scratch (ie growing 20 tripple flasks of cells to get 2 L of conditioned media to start my purification again....groan 
)
Hi again,
Hopefully a high glycerol content will help.
Also, when I purify proteins through a series of columns (or a single column)
I always check to see if there is actually any protein in the elutions.
This can be done with Bradford reagent in 96 well plates (just see if it turns blue).
It's possible that all the Wnt that you are looking for is in the first set of eluants anyways.
But check them all to see which ones have proteins.
I only run the eluants containing protein on gels.
Good luck.
)We have had that happen every now and then, and usually it was caused either by too much cell lysate, or incorrectly prepared sample buffer. I'm not sure what exactly caused the problems in each case (probably DNA in the first case). Make some more loading buffer 
I am doing protein purification of Wnt3a. I ran my protein sample on a superdex 200 prepgrage 10/30 column and collected elutions in 1X PBS, 1% CHAPS (a detergent). I wanted to run all of my elutions on a gel and coomassie stain it to figure out which elutions have protein. I took some of my elutions and added 5X laemmeli sample buffer to the proper 1X concentration. and i started loading them on a 10% acrylamide gel and i was able to load elutions 1-14 fine. Then i started to try to load elutions 15-26 and they would just float out of the well. I made sure I had added 1X SDS running buffer to the chamber and got a new gel. Also tried using a 5X sample buffer that someone else had made to make sure that the sample buffer wasn't the problem and again the samples would just float out of the well. No one in the lab can explain why this is happening. We think that there is something in my elutions that is causing the problem but aren't sure what it could be. It should also be noted that I ran another aliquot of this sample run through the same type of column but in a different lab on a different purification machine and was able to run it on a gel and coomassie stain it.
density of samples is equal or higher than of running buffer; may be too much dilution of 5x sample buffer; add additional glycerol to the sample buffer;
shorten your question for convenience
Just an aside, but in response to the "shorten your question for convenience" comment,
I have to say, it was a breath of fresh air on this web site to read an entire paragraph
that was written intelligibly and grammatically correct. Congratulations.
AND no offense meant to "The bearer". It was just an observation.
I wanted to post that i used a different recipe for a 5X high density sample buffer that had 50% glycerol instead of 20% glycerol and did not have a problem with my sample floating out of the well! 