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Dot Blot Assay - (Aug/13/2009 )

Hi all,

When can you apply the dot blot assay and why would someone chose this assay over a western blot to detect proteins?

Thanks

Pmaj

-pmaj-

Typically a dot blot is used to optimize a Western Blot. Because every Western blot involves a combination of antibody and antigen interactions, no one antibody concentration exists and optimization is essential. Appropriate primary and secondary antibody concentrations depend on each antibody's specific activity and specificity for its antigen, as well as the amount of antigen present in the sample. The specific activity of secondary antibodies varies among manufacturers. Re-optimization is necessary when one or more of the experimental variables such as the antigen, primary or secondary antibody, or substrate is changed.

Concentrations of the antigen, primary and secondary antibodies that are too high or too low can cause a variety of undesirable results. The following list includes possible results from a Western blot that would require optimization of antigen/antibody concentrations:
Weak signal
Signal fades quickly
No signal with no background
High background, white bands (reversed image)
Orange or brown spots on the membrane
Nonspecific bands
Diffuse bands
Blotchy or speckled background
The optimal antigen and antibody concentrations can be determined by performing a Western blot with varying concentrations. An easier and quicker method is to perform a dot blot. I usually use this method so I don't have to run a gel and transfer to a membrane everytime I am trying to troubleshoot my Westerns. There is a real good procedure for optimization of antibody/antigen concentrations at http://www.piercenet.com/files/TR0024-Opti...b-dilutions.pdf. A dot blot will only show a small black dot on the membrane while a Western Blot will seperate all of the proteins and only show a black spot for the protein that is being detected, a dot blot can't show if the correct protein is being recongnized or if it is a non-specific band.

-AVEA-

AVEA on Aug 13 2009, 01:58 PM said:

Typically a dot blot is used to optimize a Western Blot. Because every Western blot involves a combination of antibody and antigen interactions, no one antibody concentration exists and optimization is essential. Appropriate primary and secondary antibody concentrations depend on each antibody's specific activity and specificity for its antigen, as well as the amount of antigen present in the sample. The specific activity of secondary antibodies varies among manufacturers. Re-optimization is necessary when one or more of the experimental variables such as the antigen, primary or secondary antibody, or substrate is changed.

Concentrations of the antigen, primary and secondary antibodies that are too high or too low can cause a variety of undesirable results. The following list includes possible results from a Western blot that would require optimization of antigen/antibody concentrations:
Weak signal
Signal fades quickly
No signal with no background
High background, white bands (reversed image)
Orange or brown spots on the membrane
Nonspecific bands
Diffuse bands
Blotchy or speckled background
The optimal antigen and antibody concentrations can be determined by performing a Western blot with varying concentrations. An easier and quicker method is to perform a dot blot. I usually use this method so I don't have to run a gel and transfer to a membrane everytime I am trying to troubleshoot my Westerns. There is a real good procedure for optimization of antibody/antigen concentrations at http://www.piercenet.com/files/TR0024-Opti...b-dilutions.pdf. A dot blot will only show a small black dot on the membrane while a Western Blot will seperate all of the proteins and only show a black spot for the protein that is being detected, a dot blot can't show if the correct protein is being recongnized or if it is a non-specific band.


Thanks a bunch Avea for the information!

Bests,

Pmaj

-pmaj-