In the process of purification we use ammonium sulphate fractionation and then subject the samples to dialysis to remove the salt and carry on further processing like gel filtration, affinity etc. to achieve several fold purification and then PAGE.
If the said salt is present it gives diffused bands rather than the desired sharp ones. In this regard I want know how can complete dialysis be checked, and also if there is any better method not involving this ammonium sulphate fractionation.
You can always skip the precipitation. Depends, whether you use it also for concentration or only fractionation?
The completeness of dialysis can be checked by conductometer. Just compare fresh buffer with the one from dialysis. Other, probably easier way is to use Nessler solution to check for presence of ammonia.
But if you do PAGE after several other chromatography steps, the original ammonium sulphate should not be of concern, rather some other salt, maybe from the affinity chromatography? Depends, what is your procedure.
Thank you very much T.Hluska Sir.....
what is the alternative if i skip precipitation, actually after doing gel filtration (sephacryl S100-HR, column), i did PAGE to the fractions with highest activity and protein content but every time got diffused bands, event though there are clear signs of single band of some of the fractions, what can be the possible cause? please help....
Depends what's your aim. If only to partially purificate, just skip it and go to the next next. If you want to concentrate, you can use ultrafiltration or some chromatography, where your protein binds.
Are you sure that your PAGE is OK? If you get smear in all samples, that seems rather like problem with PAGE. How do you prepare the samples just before PAGE? What do they contain?
i started with 60 mirogram/well , got smear then lowered to 25 microgram( in a sequential manner) only to get diffused band(diffused in 1/2 cm area). i follow 4:1 sample to dye ratio( in microlitres). Well as far as PAGE is concerned i think there is nothing wrong with it, because we are getting good results with other samples...
Thank You Gallardo Sir very much .....
I will try this for sure,feeling so privileged.... so kind of you Sir...
Thank you once again.
ionex is not very suitable if Sheraz wants to desalt the sample. It would be better to go directly to the gel filtration chromatography.
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