best and simple method to purify protein - (Aug/03/2010 )
I'm new here ..i hope all of you can help me..i'm bachelor degree student and need to start my final year project.my topic is protein profiling of Pleurotus sajor-caju...i'm looking for the best method how to purify that mushroom.i only have another 1 month to finish up my project ..
It's not that simple. What do you mean by "the best"? Quickest? Cheapest? Most advanced? Sure, I can swamp you with names and acronyms of "the best" techniques but it won't make sense to you if you don't have this kind of equipment in your lab and skills to use it. Please, clarify and then we may be able to help you.
yeah give us ur limitations as well as requirements!!! we might able to work out sumthing then... but purification of a protein in one month!!
seems tricky.. u can partially purify it though by doing a TCA precipitation and then may be running it thru a column (IEX, GFC, RP etc!!)
actually i already have method:
step 1: fruiting bodies of mushroom will be blended with TRIS buffer
step 2:homogenize the sample then sieve through muslin cloth
step 3:ammonium sulfate precipitation
step 4:centrifuge and take pellet
step 6:column chromatography
In your opinion,is my method work??with this method can i purified protein in that mushroom
thank 4 ur respond i'm really appreciate it
Step 1. Add some protease inhibitors (eg. "complete" from Roche). You must also consider pH and ionic strength of buffer.
Step 2. Centrifuge after (or instead of) sieving eg. 10~15 thousands g for 15~60 minutes to completely remove any debris.
Step 3. What saturation are you planning to use?
Step 6. That's pretty generic term. What kind of chromatography? Gel filtration, ion exchange, hydrophobic interaction?
10q for ur suggestion..
actually last week 1 did ammonium sulfate precipitation with 50 percent saturation but my sample did not have any changes n not precipitate..i thought mybe my method is wrong..in ur opinion what saturation are suitable??
usually i use gel filtration chromatography but take a time to finished up...
50% saturation may not precipitate all of the proteins. I would use 100% saturation (to do that you would have to use solid ammonium sulphate - not solution) or other kind of precipitation - eg. TCA, ethanol, acetone.
If you're going to use gel filtration you may skip dialysis.
10q for ur suggestion..
i already ask lab assist n they said no protease inhibitor..is it ok if i do not use protease inhibitor?
They don't have any or they don't want you to use it for some strange reason?
Not using protease inhibitors you risk losing some of the proteins.
At least perform all activities in low temperatures (4 degC). (And use sodium azide and/or merthiolate during exctraction.)
thanks 4 ur suggestion
my project in progress...
if i want to do protein profiling its necessary to do lowry assay?