problem with amonium sulfate precipitation - What should i do ? (Apr/17/2004 )
Hi again,
Well, you want to precipitate all proteins in your solution. At 4M AS 90++% of your proteins precipitate. You need to add solid AS to a final concetration of 4M, whether it's 1ml or 1L you are playing with.
REMEMBER: As you add AS, the volume will increase, so you will need more AS than you think. Use the following formula:
g= [533(M2-M1)]/ (4.05-0.3M2), that's grams per LITER of solution
g: grams of AS
M1: initial AS concentration (more likely to be 0 unless you've added any AS)
M2: final AS concentration (usually 4M)
Also, sometimes not all of the AS gets resuspended, so if you end up with little bit of solid AS at the bottom of your tube simply add some water and allow to mix for a few minutes and repeat if solid AS is still present.
Centrifuge at higher speeds, try 19k rpm.
Good luck!
Also- you cannot use filtration to bring your enzyme volume down because media salts will increase in concentration and most probably kill the activity of the amylase before you can use diluents. Dialysis is the preferred option.
Good Luck again and let us know if you need further assistance. Write a private note to dstf@doctor.com and one of our staff enzymologists will stay with you via email 24 hours a day until you complete your practical work. Don't worry- you are in the right place and all the right experts have read your note and understood your problem.
Ultrafiltration is the best method for concentrating large volumes. Dialysis is a bit cumbersome at this level. During ultrafiltration the excess liquid with the salts escape throught the membrane. Commonly used membranes porosity rang from 3 kDa to 30 kDa. Even in 3 KDa membrane the salts escape leaving the concentrate unaffected.
One problem of low activity after ultrafiltration might be due to choice of wrong membrane. One of my enzyme has a theoretical weight of 66 KDa but passes thorugh 30 KDa cut off membrane. There pores sizes are applicable only for shperical shaped enzymes. try filtration with lower cut off membranes.
Another problem duirng ultrafiltraion, is the stirrer speed. Keep it as low as possible; otherwise it may whip the protein inot a foam and denature it.
Thirdly, use nitrogen pressure and not oxygen pressure to force the filtration. Some proteins are susceptible to oxidative stress.
Good Luck
Sharath.B
Research Fellow,
National Chemical Laboratory,
India