best and simple method to purify protein - (Aug/03/2010 )
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
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.
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
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.
K.B. on Mon Aug 16 17:28:19 2010 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..
You determine MW of your proteins with gel filtration or SDS-PAGE, using standard protein mix for calibration. If you notice that proteins are not resolving properly, you need to change chromatography gel type or electrophoretic gel percentage.
hi..
i have another question..
can i run my gel filtration manually without fractional collector??
if i run manually should i calculate every drop of the solution???
tQ
Yes, you can, but it would be very boring.
If you don't want to count drops, you can measure flow (with graduated cylinder and stopwatch) and then use timer or stopwatch to collect specific amounts based on time. Then of course you have to measure UV absorbance at 280nm for every fraction...
K.B. on Fri Aug 27 18:44:33 2010 said:
Yes, you can, but it would be very boring.
If you don't want to count drops, you can measure flow (with graduated cylinder and stopwatch) and then use timer or stopwatch to collect specific amounts based on time. Then of course you have to measure UV absorbance at 280nm for every fraction...
TQ...
time to collect the specific amount of every fraction is important or not??if i want to measure uv i should use uv-vis spectrophotometer and quartz cuvettes??
hav a nice day
intan on Sat Aug 28 12:49:17 2010 said:
time to collect the specific amount of every fraction is important or not??if i want to measure uv i should use uv-vis spectrophotometer and quartz cuvettes??
in general, you want to collect equal sized fractions (there are special cases where you don't ). so, collection by time, drops or volume is important.
and, yes, quartz cuvettes are preferred for reading in the uv spectrum (some plastic cuvettes are also uv rated).