why my freshly prepared 30% Acrylamide solution has precipitate? - (Mar/25/2007 )
Hi all,
recently I have a problem with the SDS-PAGE. I want to use 8% SDS-PAGE and western blot to detect a 125KDa glycosylated protein.
Before, I don't have any problem with seeing a clear band (although it is highly glycosylated), but recently I cannot get the same clear result! The bands always ran much broader like a smear. It seems my SDS-PAGE gel has some problem, so I tried the new TEMED, freshly prepared APS, and also prepared the fresh 30% acrylamide solution (with 0.8% bis-acrylamide). I found the acrylamide solution CANNOT fully dissolve after even 10 hours!!!!!! (I THINK IT SHOULD BE VERY EASY TO GET A CLEAR SOLUTION) So I filtered it, and do the Western Blot again, the bands are still not clear...... 
I checked the old acrylamide solution, and I found it has precipitates now! 
Anyone once met the same problem? Should Acrylamide solution have precipitates? Do you have any suggestions?
Thanks a lot in advance!!!!!
Sorva (Sad, broken-hearted but still keep a little bit hope)
acrylamide monomers solution shouldnt precipitate. My advice is to make a new monomer solution. Don't keep too long for monomer solution. It might be contaminated.
Good luck!
Thanks for your reply!
I actually did prepare a new solution, but I found it cannot fully dissolved even after 10 hours!( some particle like stuff in the solution) Do you think it is possible that the Acrylamide or Bis-acrylamide powder has some problem?
Thanks a lot!
I think your "problem" is the Bis-Acrylamide. Every time I made 30% acryl-bis, I had to filter the solution before using it, to avoid clogging in the gels. The proportion of bis that do not dissolve should be really small though. But it is normal.
And as for precipitation, I have never encountered this problem. DO you protect your solution from light?
Yes I protect the solution from light by covering the bottle with foil, and store at 4C.
Do you mean the bis-acrylamide is not easy to dissolve or it is just saturated? I use 0.8% for bis-.
And the precipitate which cannot dissolve is brown in color.
And as for precipitation, I have never encountered this problem. DO you protect your solution from light?
Brown?!
It is not what I thought then! I meant the bis do not dissolve completely. It does dissolve at about 99% though. But a brown precipitate... I have never seen anything like this! (in bis-acryl solutions 
)
how old is your acrylamide powder? I have seen yellow in solutions with less purified acrylamide stocks (cleared with decolorizing carbon and filtered with whatman #1 then 3.0 um millipore).
your acrylamide powder stock is either contaminated or decomposed. try someone else's.
the smearing is most likely caused by incomplete polymerization of your gel caused by poor acrylamide quality.
your acrylamide powder stock is either contaminated or decomposed. try someone else's.
the smearing is most likely caused by incomplete polymerization of your gel caused by poor acrylamide quality.
and high content of acrylic acid which binds to a proteins so one of the possibilities - incubate your stock with Amberlite MB150 Resin ( Supelco) to remove acrylic acid and then store you Stock with these beads. May be it help!
but, if your acrylamide is this badly decomposed then you won't know your concentration. if you want to salvage the acrylamide stock then you will have to recrystallize the acrylamide after clearing the acrylic acid.
but, if your acrylamide is this badly decomposed then you won't know your concentration. if you want to salvage the acrylamide stock then you will have to recrystallize the acrylamide after clearing the acrylic acid.
Well, if acrylamide so dirty I think the simplest way is to buy one more package. Recrystallization of acrylamide seems too harsh procedure in lab is'nt it?