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Western Blot with fixed cells

Western Blot Fixation

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5 replies to this topic

#1 Tabaluga

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Posted 28 May 2013 - 01:04 PM

Hi people,

is it possible to do a WB from cells that were fixed before (Cytoperm/Cytofix, contains PFA)? I am currently looking at literature and searching the internet on this topic, but I still wanted to ask you bioforumers for your opinion/advice/experience ! Posted Image

Thanks !

EDIT: Would an ELISA be a better alternative in this case ?

Edited by Tabaluga, 28 May 2013 - 03:06 PM.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#2 Danbas28

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Posted 29 May 2013 - 07:23 AM

I don't think it would be possible after fixation ... instead they will be fine for FACS analysis eventually

#3 Tabaluga

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Posted 29 May 2013 - 02:16 PM

Subsequent FACS analysis is not possible in this case, which is why we wanted to do a Western...

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#4 Tabaluga

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Posted 30 May 2013 - 11:05 AM

anyone else an opinion ?

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#5 doxorubicin

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Posted 30 May 2013 - 12:49 PM

One paper that might be helpful:
[Cancer Research 65, 473-482, January 15, 2005]

In it they fixed with 3.7% HCHO, 30 minutes and permeabilized with
0.1% Triton X-100 before sorting the cells, and then just made their
western lysates directly in SDS Sample buffer.

I've personally tried various fixation techniques prior to western blot, and most results are pretty horrible but depended on the protein.

Why do you need to do westerns? Can you do immunostaining? What about RNAscope technology?

#6 Tabaluga

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Posted 04 June 2013 - 03:18 PM

Thanks for the reply, doxorubicin !
Hm, with immunostaining it's not really quantitative, I suppose... and RNAScope is rather for tissue sections I think ? Had been considering ELISA as an alternative though.

Edited by Tabaluga, 04 June 2013 - 03:20 PM.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 






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