Western Blot

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Thu Oct 11 08:03:41 EST 2007


Am 10.10.2007, 13:50 Uhr, schrieb WS <novalidaddress from nurfuerspam.de>:

> On Oct 10, 1:09 pm, "Sudheendra Rao N R" <sudhe... from gmail.com> wrote:
> Dear Sudheendra,


>
> formaldehyde reacts reversibly with amino groups (eg lysines) and may
> interfere with epitope recognition. When you include some amino- or
> ammonium compounds (eg NH4HCO3) in excess in your sample prep, they
> should scavenge the formaldehyde and it should work. Maybe even the
> glycin in your running buffer is enough.

No, the Schiff base formation you are referring to is only one of the  
possible reactions of formaldehyde with proteins. In addition, you get  
cross-linking via immonium-cations
                                    - H_2O          + R'-NH_2
R-NH_2 + H_2C=O ----> R-NH-CH_2-OH ----> R-N^+H=CH_2 ----> R-NH-CH_2-NH-R'
(For non-TeXnicians: _ and ^ indicate sub- and superscripts, respectively)

and via the Mannich reaction (cross-linking between amino groups and  
tyrosine rings). These are not (easily) reversible. Although formaldehyde  
has only one functional group, it should be regarded as bifunctional  
cross-linker.

To OP: If you want to use fixed tissue for Western blotting, fix in  
alcohol, acetone or heavy metals (e.g. Zenker's solution, SuSa). The  
latter ones you may be able to use for perfusion-fixing (after a thorough  
rinse with PBS, of course).


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