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best and simple method to purify protein


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38 replies to this topic

#16 K.B.

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Posted 17 August 2010 - 12:35 AM

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.

#17 intan

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Posted 27 August 2010 - 04:42 AM

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
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#18 K.B.

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Posted 27 August 2010 - 10:44 AM

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...

#19 intan

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Posted 28 August 2010 - 04:49 AM

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
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#20 mdfenko

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Posted 29 August 2010 - 08:53 PM

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).
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#21 intan

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Posted 01 September 2010 - 07:11 AM

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???
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#22 K.B.

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Posted 01 September 2010 - 01:56 PM

both (in separate wells of course), and don't forget about molecular weight marker

#23 intan

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Posted 02 September 2010 - 06:15 AM

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...... :)
if i don't know something then i will ask others to help me and make me understand

#24 mdfenko

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Posted 02 September 2010 - 08:20 PM

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).
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#25 intan

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Posted 21 September 2010 - 11:56 AM

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... :blink:
what should i do???
if i don't know something then i will ask others to help me and make me understand

#26 mdfenko

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Posted 22 September 2010 - 06:23 AM

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.
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#27 intan

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Posted 22 September 2010 - 06:14 PM

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???? :huh:

thanks in advance :)

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#28 mdfenko

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Posted 24 September 2010 - 07:34 AM

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.
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#29 intan

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Posted 25 September 2010 - 09:30 AM


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 :(
if i don't know something then i will ask others to help me and make me understand

#30 mdfenko

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Posted 28 September 2010 - 12:15 PM

not at all. you can make out the bands in your samples.
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