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"weber & osborne" and neville's SDS-PAGE method


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#1 rick112

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Posted 17 December 2009 - 11:30 PM

Hi

could any one please provide me with protocol for neville's SDS-PAGE method and weber & osborne SDS-PAGE method?? heard that some times they give sharper bands than laemmli system (this is what we are using in our lab..so curious to give it a try...

thanks a lot

#2 mdfenko

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Posted 18 December 2009 - 11:19 AM

here are the original jbc papers.

i have two versions of weber and osborn formulations, one using a phosphate buffer, as in the paper, and the other using a tris buffer.

we routinely use neville, but, we use a modification. we use a 5-15% acrylamide gradient (it showed better resolution of our proteins than the equivalent laemmli gradient).

if you want, i can post the formulations as we use them (i'll have to type them up, first) but you should be able to get the formulations from the papers.

by the way, sorry it took so long to get this to you, rick112, i was briefly sidetracked.

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#3 rick112

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Posted 18 December 2009 - 11:54 PM

thanks for the protocol

will give it a try next week..and let all of you know the results...(hope they trun out 2 be good... :lol: )

#4 rick112

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Posted 24 February 2010 - 12:57 AM

hi

i was thinking of running the "weber and osborn" SDS-PAGE gel based on above mentioned article...there is no mention of stacking in the article, in case i want to use "Stacking" for my gel could anyone tell me what 'gel buffer' i have to use.
is it the same buffer as separating only with ph difference????

mdfenko...your help will be highly useful

#5 mdfenko

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Posted 24 February 2010 - 09:48 AM

when we ran weber and osborn gels, we ran cylindrical (tube) gels. we did not use stacking gel. with tube gels we normally limited the height of sample load to keep the bands as sharp as possible.

you could design a stacking gel very easily. make the gel ~4% and pH somewhere from 6.1-6.8 using sodium phosphate buffer.
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#6 Beggolina

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Posted 01 May 2014 - 10:19 AM

Hi, I know this is a very old conversation but Im trying to do use the weber osborn gels to look at protein oligomerization.

 

Tried pubmed and no luck finding someone that describes the technique a bit more. However I cant even get the protein to enter the gel (with the NaH2SO4 buffer). Cant even separate a marker on it.  I have the original paper and have done accordingly. Any suggestions to what Ive could have done wrong. Im doing flat gels.

 

I noticed that in the paper they say to make a 10% acrylamide solution, however its 22.2%? 22.2 g in 100 ml. So is it really 10% or is it something else. Ive done both according to protocol in paper and a proper 10% gel. No stacking. I need a low % gel in order to see the range from monomer to hexamer of my protein.

 

What voltage did you run the gel at and for how long? 

 

Thankful for tips and tricks smile.png


Edited by Beggolina, 01 May 2014 - 10:20 AM.


#7 mdfenko

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Posted 02 May 2014 - 04:02 AM

the gel itself is 10%, the stock is higher.

 

give us the complete protocol that you are following and then we can make comments.


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