15 replies to this topic

[amir1979]

dear  all
I have produced  alpha amylase enzyme with Bacillus licheniformis
in a volume of 2 liters . Assay on alpha amylase activity has been done and i am sure that it has amylase activity.
Now i want to concentrate my enzyme with Amonium sulfate precipitation and compare it with ultra filtration ,but  in three assays that have been done till now i have nt  aquire any precipitate in 85% of saturation and also any pellete .
as i persume i should have at least 1 mililiter  precipitant out of my 10-2o ml of my sample but unfortunately i dont have any precipitant .    
i am very nervouse cause i should finish my practical work up to two month later . what should i do for this problem . what do you suggest  ?
thanks in advance

amir golmohammadi
Iran

[phdconsult]

Amir

You are not getting any precipitate with ammonium sulfate because the overall protein concentration in the culture supernatant is low. I suggest you first bring down the effective volume of your culture supernatant. This can be done in a small batch of 100 ml. Remove 100 ml of the amylase containing supernatant and dialyze against 1 L 25mM Tris buffer containing 10 mM MgCl2 and glycerol at 50% v/v at 4C for 10 h. This will bring down the volume of your filtrate from 100 ml to 34 ml. Subject this dialysate to precipitation with ammonium sulfate at 4C and the process must be carried out for a total time of 48h. You will see a precipitate- it will be light and flaky but the active enzyme will be present in the pellet because amylases are quite stable.

Good luck and God Bless

[phdconsult]

Amir

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.

[Antigen]

Amir,
Ammonium sulfate precipitation works well for relatively concentrated protein solutions only. If you have less than 0.1 mg/ml - it will be difficult to concentrate, try up to 90% and overnight in cold-room. Also this kind of precipitation works better with hydrophobic proteins and with MW more than 20kDa. I would try cold aceton or ethanol.

[Shubenok]

For best precipitation pH must be lower than protein pI, cause sulphat ion interact with positive charged groups, making protein more compact.
If it is still important for you

Edited by Shubenok, 25 May 2004 - 09:51 AM.

[InvisibleSurfer]

Hi all,

Regarding am.sulfate ppt., I had the impression that at 4M concentration, ALL proteins precipitate...

[phdconsult]

InvisibleSurfer, on Jun 14 2004, 05:07 AM, said:

Hi all,

Regarding am.sulfate ppt., I had the impression that at 4M concentration, ALL proteins precipitate...

That is true provided the protein is occupying salt 'space'. A low concentration protein can coexist with that concentration of ammonium sulfate because their individual solvations have not been interfered with. A very good point  though and is used in Biochemical engineering for seeding fermentation products by adding denatured papain.

[Proteinjunkie]

I have a different problem with AS precipitation. When I add water not all the protein resuspends after the A.S. What am I doing wrong?

The volume of water I add is the same as the starting volume of the protein sample (2ml) prior to adding the A.S. It doesn't resuspend everything and I am still left with a white precipitate. Any hints? Should I be adding a buffer and not water?

The starting concentration of the protein solution is 5mg/ml and I perform the precipitation by slowly adding A.S to the protein solution which is mixing at the time. I then leave it stirring at room temp for 10 minutes before centrifugation at 15,000g for 10 minutes at 4oC. I resuspend at room temperature. The A.S solution I made was done by adding A.S to water (boiling) until no more went into solution. I cooled it down to 4oC and spun out the remaining undisolved A.S. Is that the right way of doing that? I am assuming that this is a 100% solution.

Proteinjunkie

Edited by Proteinjunkie, 04 August 2004 - 07:17 PM.

[InvisibleSurfer]

I think you are making a big mistake! You should add SOLID ammonium sulfate at whatever concentration you need it and then add a little bit of water ONLY if you see any undisolved ammonium sulfate at the bottom of the beaker.

Remember to add it slowly and give it a good 2 hours to mix with your protein solution.

[Proteinjunkie]

What if I am working with only 2mls....? Same advice?

[InvisibleSurfer]

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!

[sharath]

phdconsult, on Apr 17 2004, 07:40 AM, said:

Amir

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

[sudhakar mutyala]

Hi
Ofcourse majority of protiens went into the precipitation by adding saturated ammonium sulfate or salt but you need to look or optimize at which salt concentration or precentage your desired protien will go into the precipitaion.

there is no need to concentrate before you go for amm sul precipitaion, Salting out will anyway reduces the water availabity to the protein , and it is wonderful technique to concetrate the dilute proteins.

Better add solid salt directly to the sample and check , avoid local concentration of the salt and stirr slowly at cold conditions.
Amir can try with little excess volume. with 2 ml you wont get a good ppt and ofcourse it depends on the protein concentration in the sample

Regards
Sudhakar

InvisibleSurfer, on Aug 10 2004, 02:40 AM, said:

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!

[lola]

well i dont have much idea regarding this....i think something in your body is low which is causing all these things....but yes take advice from some expert...

[shane]

A  reduced   engrossment  protein can coexist with that  engrossment  of ammonium sulfate because their  one-by-one  solvations have not been  hindered  with. A very good  issue  though and is  utilized  in Biochemical  technology  for seeding fermentation  goods  by  supplementing  denatured papain.