Protocol Online logo
Top : Forum Archives: : SDS-PAGE and Western Blotting

White precipitation in stacking gel - (Oct/29/2008 )

Pages: 1 2 Next

Hi,

I have been running westerns for a while now and never encountered this problem. Today when I was making my gels I kept getting a white precipitation in the stacking gel. The ingredients for my resolving gel and stacking gel are the same, just different quantities. The only big difference is that the resolving gel has 1.5M Tris HCl & the stacking has 0.5M Tris HCl. So I thought maybe this was the cause. However, I got some fresh 0.5M from a colleague & got the precipitate again. If its some other ingredient I'm using I would expect to see the precipitate in the resolving gel also but I only got in the stacking gel. After 3 attempts I gave up. Someone in the lab suggested it was the SDS precipitating out of solution but again I use the same SDS solution in the resolving gel & the solution itself looks fine, there's no presence of a precipitate.

Any one any idea's on what it might be???

Gels contain: Water, Tris HCl, Acrylamide, APS, 10% SDS & TEMED.

Thanks in advance!

-moorele-

Sounds like something external making problems. What do you use on your stacking gel to keep it airtight after pouring? Do you overlay with water or something else? Perhaps there is something, which interferes with your stacking gel solution.

-biomaus-

Do you pour your stacking in a cold room?

Could it be that the SDS is precipitating?

-Madrius-

I poured the stacking gel on the bench like always... Have never had a problem before. The weather is quite cold here at the moment but the temperature in the lab isnt much colder than normal. Someone else in the lab suggested it could be the SDS precipitating due to the colder weather but surely this would have happened in the resolving gel too as it also contains SDS.

I dont do anything to keep the stacking gel airtight after pouring. I pour it on top of the resolving gel and put the comb in and leave it to set on the bench. I've done this hundreds of times before but never had a problem with it setting or anything precipitating.

-moorele-

may be some residue chemical on the comb or the glass plate.

-Minnie Mouse-

are you seeing a precipitate or is the stacking gel turning milky?

the stacking gel can turn milky if you have a higher concentration of bisacrylamide.

do you use separate acrylamide solutions for the stacking and running gels?

what are the concentrations of acrylamide and bisacrylamide?

-mdfenko-

QUOTE (mdfenko @ Oct 30 2008, 02:36 AM)
are you seeing a precipitate or is the stacking gel turning milky?

the stacking gel can turn milky if you have a higher concentration of bisacrylamide.

do you use separate acrylamide solutions for the stacking and running gels?

what are the concentrations of acrylamide and bisacrylamide?



I would say its a precipitate as it settles to the bottom.

I use the same acrylamide for both the resolving & stacking gels. & the concentration of the acrylamide is 30%. Only difference is the resolving gel is a 12% gel so 8mls of 30% acrylamide is used & the stacking gel is a 4% gel so only 1.33mls of 30% is used.

-moorele-

a long shot, when you prepared the 0.5M tris, did you overshoot the pH and return it with koh?

kds is much less soluble than sds and will form a white precipitate.

other cations will also form precipitates with sds, maybe something else is contaminated or insufficiently rinsed after cleaning (glassware, plates).

-mdfenko-

I'm guessing a pH problem as well: you shouldn't use bases when preparing tris solutions, it damages the buffering capacity of the tris.

-bob1-

QUOTE (bob1 @ Oct 30 2008, 11:24 PM)
I'm guessing a pH problem as well: you shouldn't use bases when preparing tris solutions, it damages the buffering capacity of the tris.


Sorry for taking so long to reply I was out last week getting my wisdom teeth out sad.gif

Have just made a fresh gel now with all the same solutions as before. Have done nothing different, havent changed any solutions & it has set perfectly fine. No precipitate! A bit bizzare! Only thing I can think of is perhaps glassware wasn't rinsed well as suggested above or the fact that it was a bit colder than usual last time & the SDS precipitated out of solution! Either that or my western voodoo went with my wisdom teeth biggrin.gif

-moorele-

Pages: 1 2 Next