Hello Sammer-ul- Hassan,
The principle of Western blotting using a blot in my opinion has to do with having proteins immobilized and then probing them with a specific antibody to detect if your protein of interest is present. Now, in a gel, the protein is not immobilized enough, it can diffuse through the gel pores and spread out. This would make antibody detection a mess and you would end up with smears that mean nothing since your protein may have diffused wherever.....even at low temperatures.
In addition to this, since the gels are not prepared in absolutely protein free environment, the probing antibody may get lost through non-specific binding. One can't quite block a gel as efficiently due to its porosity....unless things are modified to chemically interact and stick to poly acrylamide. Thus, a membrane/blot is used to achieve protein in a fixed location, since the mesh of the blot is not as diffusion friendly for large molecules such as proteins. Hope this helps.
Regards
Hello Sammer-ul- Hassan,
The principle of Western blotting using a blot in my opinion has to do with having proteins immobilized and then probing them with a specific antibody to detect if your protein of interest is present. Now, in a gel, the protein is not immobilized enough, it can diffuse through the gel pores and spread out. This would make antibody detection a mess and you would end up with smears that mean nothing since your protein may have diffused wherever.....even at low temperatures.
In addition to this, since the gels are not prepared in absolutely protein free environment, the probing antibody may get lost through non-specific binding. One can't quite block a gel as efficiently due to its porosity....unless things are modified to chemically interact and stick to poly acrylamide. Thus, a membrane/blot is used to achieve protein in a fixed location, since the mesh of the blot is not as diffusion friendly for large molecules such as proteins. Hope this helps.
Regards
in addition to the above the rate of reaction of antibody and antigen depends on the concentration of the reagents ...higher concentration equates to quicker reaction so immobilising antigen on the membrane increases its concentration so speeds up the reaction as well as allowing storing of the membrane for further probing with different antibodies which would be difficult with gels even dried acrylamide gels
Protocols do exist that describe In-Gel Westerns and are supported by companies. For instance, we have a LI-COR Odyssey Imaging System in our lab and the manual lists In-Gel Westerns as one of its applications. Due to the nature of fluorescently labelled secondary antibodies In-Gel Westerns are feasible on the LI-COR system. You probably can't do an ECL based detection with gels.
The problem with these In-Gel Westerns are the excessive amount of antibody that you will need to incubate your gel with. Also, the signal intensity will be weaker and you may not be able to recycle your antibody back.
Thanks very much for your answers. I was planning for microchip western blotting and as suggested by Thomas U.R. Gantert, the LI-Color systems provide at least an example that western can be done in a gel.
Thanks to Aparajita Lahree and Paul Rutland for letting me know the possible problems for In-gel westerns. I really appreciate the answers. In my case, the gel thickness will be 0.3-0.4 mm and the the antibody can be in high concentration which I hope the reaction can be faster and might require small portions of antibody solution on the gel (By estimating the size of the antigen from the markers).
there might be some scope for drying the gel onto a plastic base ( film ) which woulf reduce the thickness and, therefore , the amount of reagen needed and stop both diffusion of the sample and, hopefully, keep the background low and make handling more robust. good luck,
Paul
- Badges
- Science method