Hi Beth, are you looking for amyloids or plasma protein aggregates?

SDS is generally considered to be the best way to solubilize protein aggregates (amyloids, not so much).

If you really want to be aggressive, I would recommend boiling the filter in SDS-PAGE reducing buffer.

Thanks Steingrimur for your quick reply.  I am looking at protein aggregates. I think I will try boiling in SDS reducing buffer as you suggest. What about reagents like Urea or Guanidinium Chloride? Other detergents, Triton? Chaps?

Hi Beth, if you want to solubilize protein aggregates and take no prisoners, then you might want to make the SDS reducing buffer with 6M urea, but I don't think that will be necessary.

Please do not use guanidinum Cl with SDS. It will precipitate. 

TX, Chaps, cholate and octyl glucoside are wimpy compared to SDS.

Very cool thanks. I definitely don't want to be wimpy.  :)

That is a good and yuge  political slogan: "No More Wimpy Detergents".

Treat it with chemical denaturants

1-Urea: 8M may or may not dissorve evetything.  The advantage is that you can use it as it is for SDS-PAGE analysis, no need to remove urea.   If you don't see proteins, or if you see some but wonther whether there are more, go to step 2

2-Use 7 M GdmCl.  In this case you will have to eliminate it for SDS=PAGE analysis.   For this, you preciptate with 10-15% TCA (trichloroacetic acid, there are standard protocols for this), whash pellet with cold acetone, add cracking buffer and load on SDS-PAGE.

In both cases, use a small volume (0.1-1 ml?), enough to cover the filter, i.e., rolled within an eppendorf tube.   

If PAGE bands for massspecking are not enough and you want the proteins soluble, you should write a grant application or enroll in a separate PhD.   Well, if you are lucky enough some proteins may be recovered soluble after 1/10 dilution or dialysis of the denaturant.

Thanks Gonzalo

I forgot, you can leave it at least an hour, increase temperature, say 40 deg, if you like (nothing will happen to the proteins in denaturants) as it may improve extraction.  Or leave it overnight.  THis is the extreme picture, maybe you get them out in 5 minutes, I don't know your proteins