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western blot


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17 replies to this topic

#1 distazio

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Posted 27 May 2009 - 02:54 AM

dear all

do u think is possible to see in western blot in the same lane 2 proteins with 4 KDa of difference?
one of 220 KDa and the pther form of 216KDa????

#2 little mouse

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Posted 27 May 2009 - 03:10 AM

I would say it's possible, I already saw such result but you need to add glycerol in the gel. Never did it myself. I have no protocol. sorry

#3 Velella

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Posted 27 May 2009 - 03:27 AM

I think it might be, if your gel is an high acrylammide-bis acrylammide % gel (15% or more)

#4 warsel

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Posted 27 May 2009 - 06:57 AM

it's difficult, but if you run the gel long enough and, as Velella suggested, use a high % gel it should be possible in principle.

people do similar things for ubiquitin which is 8.5 kD

#5 labmeiser

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Posted 27 May 2009 - 07:56 AM

I have been able to see a 4kb difference when running a gradient gel at lower speeds (For longer time). 4-20% gradient run at 80V (regular 5cmX7cm gel).

#6 mdfenko

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Posted 27 May 2009 - 08:38 AM

we separated proteins in the range you need (isoforms of myosin heavy chains) by running a 3.5-5% gradient gel with a 8-0M urea gradient.

Edited by mdfenko, 27 May 2009 - 08:39 AM.

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#7 mdfenko

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Posted 27 May 2009 - 10:03 AM

we ran 3.5-5% acrylamide with a 8-0M urea gradient gel (neville buffer formulation, not laemmli but that may work, as well) to separate myosin heavy chain isoforms. it should work for you.
talent does what it can
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#8 mdfenko

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Posted 27 May 2009 - 10:10 AM

this is the third time i am seeing the same question. you only need to post the question once. more than that becomes annoying.

as i answered twice before, a 3.5-5% acrylamide with 8-0M urea gradient, sds-page, was used to separate myosin heavy chain isoforms. try it if you wish.

Edited by mdfenko, 27 May 2009 - 10:18 AM.

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#9 HomeBrew

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Posted 27 May 2009 - 01:23 PM

mdfenko is not going nuts -- neither are you. I merged what was originally three threads asking the same question into one thread. mdfenko answered the question as posted by distazio in all three threads, and since the answers were not identical, I didn't feel I should delete any of them...

#10 distazio

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Posted 30 May 2009 - 02:56 AM

tnks

i'll try like mdfenko suggests.

#11 distazio

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Posted 30 May 2009 - 03:08 AM

could you guve me a protocol for gradient gel?

tnks

#12 mdfenko

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Posted 03 June 2009 - 12:32 PM

do you want the complete gel formulation or are you just asking how to pour a gradient?
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#13 distazio

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Posted 04 June 2009 - 02:02 AM

i think..there are commercial gel 3.5-5% gradient gel with a 8-0M urea gradient. is it true??

than...is possible and usefull trypsinize the protein and than see the difference on smaller fragments..what do you think??

#14 mdfenko

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Posted 04 June 2009 - 06:11 AM

i think..there are commercial gel 3.5-5% gradient gel with a 8-0M urea gradient. is it true??

i haven't seen them, but i haven't really looked.

then...is possible and useful to trypsinize the protein and then see the difference on smaller fragments..what do you think??

are the proteins related? will you see cross reactivity with the same antibody?

if not then you could stain, strip and restain if the proteins are in the same lane or cut the membrane and stain separately if the proteins are in different lanes.

fragmenting and then staining seems to be overkill but you could do it if you know the fragment pattern for each protein. keep in mind that the antibody will only detect the fragments that contain the epitope(s) that were used to inoculate.
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#15 distazio

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Posted 05 June 2009 - 01:56 AM

tnks a lot.

but i suppose that is not easy to do. i'm working in a genetic lab and we haven't a great experience in proteins.

but for me is important to see the two myosin protein with 3 KDa of difference in my Knock-in ES cells. but i don't know which is the best and simple system to use.

tnks very much.

mariateresa from italy




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