Hello Readers..
Today we learn something about Western blotting. I do not have an idea as to why they it is called western, but let's get an idea about blotting technique.
Western blot is a technique to detect specific protein present in any sample.
Well to identify the proteins, first they need to be separated which is carried out by gel electrophoresis. You can check it out over here.
After separation of the protein fragments, they are transferred onto nitrocellulose membrane.
Nitrocellulose membrane is used because proteins bind well to it.
There can be two methods for this transfer :-
a) This is a newer method using electricity. Voltage is applied across the gel and the fragments are pulled on to the membrane and maintain their original organisation.
b) This is a traditional method in which capillary movement of the buffer is used. This method is a bit time consuming. The nitrocellulose sheet is placed above the gel , and a weight is kept at the top. the entire stack is kept in a transfer buffer. the buffer moves up due to capillary action and brings the proteins from the gel onto the membrane in the same organisation.
The next step is probing.
Antibodies specific for the target proteins are used as a probe. This will be our primary antibody. These probes will bind to the target proteins, and any unbound antibodies are washed off. Any non specific binding, like binding of the probe to the nitrocellulose sheet is avoided by blocking.
Blocking is a step done prior to addition of the probe. they membrane is immersed into a solution of Bovine Serum Album (Rich source of Protein) in buffered Saline. the proteins in BSA bind to the nitrocellulose membrane all over where the target proteins have not bound (as in they fill up all the empty space on the membrane), thus leaving no room for the probes to bind.
After incubation post probing for 24 hrs at 4oC, a secondary antibody which will be specific to the primary antibody is added to sort of amplify the signal. Our this secondary antibody, like in ELISA will be something that produces signal like production of a colored reaction or radioactivity. Reporter enzymes are widely used for production of luminiscence. After addition of secondary antibody, the membrane is incubated for 2-4 hours at room temperature.
Next step is detection:
Detection of the target protein will vary according to the secondary probe used.
For eg.
For a colored substrate a colorimetric detection will be used.
For a radioactive label radioactivity detection is required. this can be done simply by exposing a X ray film to the membrane and it will develop showing darker spots where the label must have bound the target protein, etc.
Western blotting is a very important technique in practicals as well as exam point in view.
Hope this post helps you all learn.
Keep learning and do not forget to be amazing <3
Lots of love
-Staph.
After separation of the protein fragments, they are transferred onto nitrocellulose membrane.
Nitrocellulose membrane is used because proteins bind well to it.
There can be two methods for this transfer :-
a) This is a newer method using electricity. Voltage is applied across the gel and the fragments are pulled on to the membrane and maintain their original organisation.
b) This is a traditional method in which capillary movement of the buffer is used. This method is a bit time consuming. The nitrocellulose sheet is placed above the gel , and a weight is kept at the top. the entire stack is kept in a transfer buffer. the buffer moves up due to capillary action and brings the proteins from the gel onto the membrane in the same organisation.
The next step is probing.
Antibodies specific for the target proteins are used as a probe. This will be our primary antibody. These probes will bind to the target proteins, and any unbound antibodies are washed off. Any non specific binding, like binding of the probe to the nitrocellulose sheet is avoided by blocking.
Blocking is a step done prior to addition of the probe. they membrane is immersed into a solution of Bovine Serum Album (Rich source of Protein) in buffered Saline. the proteins in BSA bind to the nitrocellulose membrane all over where the target proteins have not bound (as in they fill up all the empty space on the membrane), thus leaving no room for the probes to bind.
After incubation post probing for 24 hrs at 4oC, a secondary antibody which will be specific to the primary antibody is added to sort of amplify the signal. Our this secondary antibody, like in ELISA will be something that produces signal like production of a colored reaction or radioactivity. Reporter enzymes are widely used for production of luminiscence. After addition of secondary antibody, the membrane is incubated for 2-4 hours at room temperature.
Next step is detection:
Detection of the target protein will vary according to the secondary probe used.
For eg.
For a colored substrate a colorimetric detection will be used.
For a radioactive label radioactivity detection is required. this can be done simply by exposing a X ray film to the membrane and it will develop showing darker spots where the label must have bound the target protein, etc.
Western blotting is a very important technique in practicals as well as exam point in view.
Hope this post helps you all learn.
Keep learning and do not forget to be amazing <3
Lots of love
-Staph.
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