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Electrophoresis of proteins from cells


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#1 electros

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Posted 05 March 2019 - 01:26 PM

Hello

 

I want to run electrophoresis for keratinocytes. These as you know, are cells that contain some proteins and they have lost most of their other cell organelles.

 

 

Is it possible to break the cell membranes so that their proteins can be moved?

Also, what else would be needed to put them on electrophoresis?

 

Thanks!

 


Edited by electros, 05 March 2019 - 01:27 PM.


#2 mdfenko

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Posted 05 March 2019 - 02:54 PM

you can break cells with detergents (eg sds, triton) or sonication or with a homogenizer (or a combination).

 

if you want to separate by size then you can run sds-page. if by charge and size then native page or urea page. if by pI then ief page. and the appropriate apparatus and power supply.


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#3 electros

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Posted 07 March 2019 - 11:32 AM

you can break cells with detergents (eg sds, triton) or sonication or with a homogenizer (or a combination).

 

if you want to separate by size then you can run sds-page. if by charge and size then native page or urea page. if by pI then ief page. and the appropriate apparatus and power supply.

 

Thanks, I think I will try to separate them in both ways, by size and then by charge.

 

Sorry I am novice and I have some questions please:

 

What do you mean by 'pl'?

Can I run the electrophoresis without breaking the cell membranes? Will it work?

Do I need to have a medium to apply the electric field? I.e. do I need to put the cells in a material and then apply electric field?

Is there a way to use a probe to apply the electric field on a specific point, rather than the whole thing?

 

Thanks!



#4 mdfenko

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Posted 07 March 2019 - 02:22 PM

pI is the isoelectric point (pH where the net charge on the protein is zero, won't migrate in an electric field) of the protein

 

not if you want to separate the proteins, plus i've never seen electrophoresis of un-lysed cells

 

there is a stationary matrix (the gel) and a mobile phase (electrophoresis buffer). the buffer, which is also in the gel matrix, completes the circuit. a sample buffer is added to the protein solution (lysed cells, in your case).

 

i'm not sure what you mean by "Is there a way to use a probe to apply the electric field on a specific point, rather than the whole thing?". you can run individual tube gels but slab gels are better for comparisons.

 

you would benefit from a handbook of electrophoresis. here are some links:

 

https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/protein-gel-electrophoresis-technical-handbook.pdf

 

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf

 

http://kirschner.med.harvard.edu/files/protocols/GE_proteinelectrophoresis.pdf

 

https://www.gbiosciences.com/image/pdfs/handbook/Protein_Electrophoresis_Handbook.pdf


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#5 bob1

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Posted 07 March 2019 - 02:51 PM

@mdfenko: That got me thinking, I've come across it before but never used it...

 

https://books.google...epage&q&f=false

 

There are references going back to the 1960's 

 

Also - for a cool assay, but removing the proteins from the cell after gel implantation: https://www.sigmaald...omet-assay.html



#6 mdfenko

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Posted 08 March 2019 - 02:46 PM

@mdfenko: That got me thinking, I've come across it before but never used it...

 

https://books.google...epage&q&f=false

 

There are references going back to the 1960's 

 

Also - for a cool assay, but removing the proteins from the cell after gel implantation: https://www.sigmaald...omet-assay.html

nice find bob. i wasn't aware of that electrophoresis method for cells. however, based on electros' original question, s/he wants to run protein electrophoresis.


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#7 bob1

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Posted 09 March 2019 - 05:41 AM

I agree that's the original intention, and it is probably substantially easier than cell electrophoresis



#8 electros

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Posted 11 March 2019 - 02:35 AM

But will the protein electrophoresis run without breaking the cell membranes?



#9 bob1

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Posted 11 March 2019 - 05:07 PM

No it won't - to get the proteins out of the cells you generally need to lyse (disrupt the cell membranes) somehow and get the proteins separate from the membranes so that they can be separated by size and/or charge. If you don't do this, then you usually can't tell much about the proteins themselves. Exactly how you get them separate from the cell and its membranes depends on which of the protein electrophoresis techniques you plan to use. 

 

I suggest you have a look around for protocols on denaturing protein electrophoresis and native protein electrophoresis. Current Protocols (journal) and Nature Protocols often have good in-depth background on these techniques and why/when you would use them. You can also go to your local library (or online) and see if you can find Molecular Cloning: a laboratory manual by Sambrook et al. It has pretty much all the techniques and background you are likely to need.



#10 mdfenko

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Posted 14 March 2019 - 06:33 AM

also, if you search the forums, then you will find formulations for various page methods that i've posted in the past (if you want to prepare your own). i would, however, suggest that if you are not going to do this on a regular basis, and even if you are, that you purchase and use precast gels and the apparatus for which they are dedicated. they have become relatively inexpensive and uniform. the apparatus, other than the power supply, is also relatively inexpensive (and, at least one of your colleagues is certain to have one).


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