best and simple method to purify protein - (Aug/03/2010 )
tQ... 
if i want to separates my protein using sds page..
what should i load in the well..my pure protein sample or my sample from chromatography???
both (in separate wells of course), and don't forget about molecular weight marker
hi...
i decide to separates the protein using sds page kit nupage novex Bis tris mini gel..
can someone explain to me what means by reduce sample and non reduced sample????
if i want to compare 2 protein should i separates them in separate gel or only in one gel?
another question about my protein sample???
how long it takes to degrades??
if i store in 4 degree Celsius for 4 weeks it can degrades or not???
Tq for helping me......
intan on Thu Sep 2 14:15:14 2010 said:
hi...
i decide to separates the protein using sds page kit nupage novex Bis tris mini gel..
can someone explain to me what means by reduce sample and non reduced sample????
if i want to compare 2 protein should i separates them in separate gel or only in one gel?
another question about my protein sample???
how long it takes to degrades??
if i store in 4 degree Celsius for 4 weeks it can degrades or not???
Tq for helping me......
reduced sample has dithiothreitol or 2-mercaptoethanol in the sample buffer to break disulfide bonds (reduce them to sulfhydryls). this allows you to break the protein up to its subunits and may also allow more complete denaturing of the subunit.
it is best to compare samples in the same gel (slab, not cylindrical gel) and, if possible, adjacent to each other.
stability of the protein depends on the protein and the conditions of storage (temperature, buffer, etc).
hi..
i already separates my protein using nupage novex bis tris mini gel 10 % 1.0mm 10 well (reduced sample ingredients)..unfortunately 
i got distorted band and my protein do not have any sharp band...only protein ladder have a clear bands...does it means my sample already contaminated and degrades??
i already did it for twice and got the same result... 
what should i do???
it would help if you would show a picture of your gel...
smears can be caused by overloading and aggregation, as well as degradation. if it smears from the proteins weight up then it may be caused by overload and/or aggregation.
mdfenko on Wed Sep 22 14:23:23 2010 said:
it would help if you would show a picture of your gel...
smears can be caused by overloading and aggregation, as well as degradation. if it smears from the proteins weight up then it may be caused by overload and/or aggregation.
my sds page pics....
if my sample become smear b'coz of overloading....i should reduce amount of sample loading or diluted it with buffer????
thanks in advance
intan on Thu Sep 23 02:14:05 2010 said:
if my sample become smear b'coz of overloading....i should reduce amount of sample loading or diluted it with buffer????
reduce the amount of sample in your load.
your pictures show samples that may not have been sufficiently denatured. do you boil your samples in loading buffer? how long? you may be creating aggregates by overboiling. you can try heating at 60-70C for 10-20 minutes instead of boiling.
mdfenko on Fri Sep 24 15:34:36 2010 said:
intan on Thu Sep 23 02:14:05 2010 said:
if my sample become smear b'coz of overloading....i should reduce amount of sample loading or diluted it with buffer????
reduce the amount of sample in your load.
your pictures show samples that may not have been sufficiently denatured. do you boil your samples in loading buffer? how long? you may be creating aggregates by overboiling. you can try heating at 60-70C for 10-20 minutes instead of boiling.
i just boil my sample (in eppendoft tube) in water bath 70c for 10 min...
actually my project due date is yesterday...that mean we not allowed to continue ours experiment anymore
if i want to use that pictures as my final result what should i explain to my supervisor???
does its mean my experiment is failed
not at all. you can make out the bands in your samples.