chemiluminescence detection/western blot

James Smith jsmith at bioch.ox.ac.uk
Tue Feb 21 08:04:27 EST 1995



On 18 Feb 1995, Shahram Mori wrote:

> janapati at ouvaxa.cats.ohiou.edu wrote:
> : I am getting high background and multiple proteins are getting labelled.  I am using monoclonal primary antibodies.  Any suggestions on blocking agent or detection method.  
> 
> I use 1:6000 or 1:7000 dilution of my 2ndry antibody and Use Gelatin to block.
> The latter can make a big difference.
> I make my blocking solution this way;
> 
> 3% Gelatin
> 1X TBS
> 2omM Tris-HCl PH 7.5
> 0.05 M NaCl
> 0.1% NaAzide
> 
> For my antibody I add 1 part above and 3 parts water and then add the relevant
> amount of antibody.
> Cheers
> --
> Shahram Mori					   _/\_
> Program in Molecular Biology			  _\  /_
> Dept. of chemistry and Biochemistry Box 3C	  \_  _/
> NMSU  Las Cruces NM				    ||
> 88003
> 

Increase the number of washes (I use PBS 0.1% Tween). Wash the membrane 
twice then wash for 3 x 20 min or 5 x 5min in the PBS solution. Do this 
after the primary and secondary steps and after you have blocked the 
membrane at the begining ( PBS/Tween with 5% Skimmed milk or I suppose 
you could use BSA).  
Another alternative is to use TBS/Tween (buffered at pH 6.8 or 
whatever the isoelectric point of IgG is!) instead of PBS/Tween (pH 7.5)

James Smith 
Dept of Biochemistry 
Oxford University
U.K.




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