Reconstitution of protein standard - (Apr/01/2009 )
Hi, I'd like to know the way u reconstitute the freeze-dried protein standard...
i got a 99% pure freeze-dried BSA powder and wanna make a standard solution with BSA conc. of 5 microgram per microlitre...
i have tried to dissolve them in lysis buffer (i weighted 0.025g BSA powder in 5ml lysis buffer), but some kind of gel like thing (i guess is the BSA) is found at the bottom of the tube. Is this a correct way to do it?
Any good idea to mimimize the error when weighting such a minute amount of BSA powder? but if reconsitute in large amount, i don't think i can use that much.
thx very much~
barnacleman on Apr 1 2009, 11:27 AM said:
i got a 99% pure freeze-dried BSA powder and wanna make a standard solution with BSA conc. of 5 microgram per microlitre...
i have tried to dissolve them in lysis buffer (i weighted 0.025g BSA powder in 5ml lysis buffer), but some kind of gel like thing (i guess is the BSA) is found at the bottom of the tube. Is this a correct way to do it?
Any good idea to mimimize the error when weighting such a minute amount of BSA powder? but if reconsitute in large amount, i don't think i can use that much.
thx very much~
Don't you have any 1 ug/ul liquid ampules of BSA around for protein assays or restriction digestions?
barnacleman on Apr 1 2009, 07:27 AM said:
i got a 99% pure freeze-dried BSA powder and wanna make a standard solution with BSA conc. of 5 microgram per microlitre...
i have tried to dissolve them in lysis buffer (i weighted 0.025g BSA powder in 5ml lysis buffer), but some kind of gel like thing (i guess is the BSA) is found at the bottom of the tube. Is this a correct way to do it?
Any good idea to mimimize the error when weighting such a minute amount of BSA powder? but if reconsitute in large amount, i don't think i can use that much.
thx very much~
BSA dissolved also comes with restriction enzyme (although I cannot remember the conc of that).
When I dissolve BSA for i.e. immunohistochemistry, I do get the gel like thing you describe, but it dissolves after leaving it on a rocker for an 10-40 mins depending on the concentration. If you just tried to dissolve it by pipetting I think simply leaving a bit will solve your problem
BSA is a very soluble protein and you can make > 200 mg/ml solutions even in water.
If I were you I would prepare a stock solution of say x10 (50 mg/ml) in water. This could then be diluted in your lysis buffer to make a standard.
Classically, the "correct" way to do this is:
1. Make an approx. 50 mg/ml BSA solution by sprinkling the powder onto the surface of the water. Let it dissolve overnight at 4 oC.
2. Filter 0.22 um to remove any undissolved material
3. Determine the concentration of the BSA by measuring the absorbance at 280 nm using a 1:100 dilution of your stock. The extinction coefficient for BSA in water is 0.667 ml mg-1 cm-1. Thus the measured A280/0.667 x 100= Your BSA concentration in mg/ml.
Hope this helps