38 replies to this topic

[intan]

dear all
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 ..
thank you

[K.B.]

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.

[Prep!]

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!!)

[intan]

hello...
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 5:dialysis
step 6:column chromatography
step 7:SDS-PAGE  

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

[K.B.]

Looks good.

Few comments:
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?

[intan]

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...

[K.B.]

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.

[intan]

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?

[K.B.]

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.)

[intan]

thanks 4 ur suggestion
my project in progress...
if i want to do protein profiling its necessary to do lowry assay?

[Chiapet874]

Adding protease inhibitors greatly protects your experimental proteins- they usually aren't too expensive and are well worth the cost in my opinion O_O

[K.B.]

intan > Lowry assay (or any other protein assay) is necessary to quantify the amount of protein in your extract so you can properly load chromatography column or electrophoresis gel.

[intan]

hi...
i'm confuse ...to run gel filtration,1st we must prepare gel media to load in column in my lab hav 3 type of bio gel p-100,p-10 and p-60..
it is based on what??size of pores??what media should i use..

TQ

[K.B.]

Google is your friend!

Bio-Gel P Instruction Manual (You should have this in your lab!)

You pick type of gel based on molecular weights of your proteins. P-100 should be OK for a start. You need to pack and calibrate your column then run your proteins. If you would have proteins with higher MW than gel can handle, you need other gel with bigger pores (in this case - Sephadex, Superdex or Sepharose). If you would have proteins with lower MW - use P-10.

[intan]

K.B., on 16 August 2010 - 09:28 AM, said:

Google is your friend!

Bio-Gel P Instruction Manual (You should have this in your lab!)

You pick type of gel based on molecular weights of your proteins. P-100 should be OK for a start. You need to pack and calibrate your column then run your proteins. If you would have proteins with higher MW than gel can handle, you need other gel with bigger pores (in this case - Sephadex, Superdex or Sepharose). If you would have proteins with lower MW - use P-10.

TQ...i already got the manual...
how can i determine my protein MW???
i'm dummies about this field..