Introduction to the principles related to the immunoblotting experiment
Introduction to the principles related to the immunoblotting experiment
Introduction and principles of Western blotting
Western blot is used to identify large antigens (usually proteins) that can interact with specific antibodies and to determine the size of the antigen. Proteins are first separated by SDS polyacrylamide gel electrophoresis and then transferred by electrophoresis to solid phase supports, which include nitrocellulose membranes, polyvinylidene difluoride (PVDF) membranes and cationic nylon membranes. Unreacted sites on the membrane are first closed to inhibit non-specific adsorption of antibodies so that the immobilized protein can interact with specific polyclonal or monoclonal antibodies. Finally, the sites are localized by radiation, colorimetric or chemiluminescent methods.
Experimental routine reagents
1. 1.0 mol/L TrisHCl (pH 6.8)
2. 1.5 mol/L TrisHCl (pH 8.8)
3. 10% SDS
4. 10% amine persulfate (APS)
7. Reduced 5X SDS Sampling Buffer
8. 10X electrophoresis buffer
9. 10X membrane transfer buffer
10. 1X Transfiltration Buffer
11. 10XTBS buffer
12. 1XTBST buffer
13. blocking buffer: 1X TBST with 5% w/v skimmed milk or 1X TBST with 2%-5% bovine serum albumin (BSA).
14. primary/secondary antibody dilution buffer: the normal dilution is 1X TBST with 5% BSA or 5% skimmed milk, see primary/secondary antibody instructions; each antibody has a fixed optimal dilution ratio.
Operating procedure
1. Determine the concentration of the gel (isolate) according to the molecular weight of the protein to be measured.
2. Lysing: Use the appropriate lysing solution and method to lyse walled cells, suspension cells, or tissue samples.
3. Run the gel: 80 volts is recommended for concentrated gels, and 120-180 volts is recommended once the sample is in the separator gel. 4.
4. After running the gel, remove any sample-free excess from the gel and pre-wet the filter paper and sponge.
5. Separated proteins are transferred to a membrane carrier.
Selection of suitable membrane
Membrane transfer: Take NC membrane as an example
Wash the NC membrane with Transfer Buffer 10 minutes to half an hour in advance.
Install the membrane transfer system according to the following structure: negative electrode - sponge - triple filter paper - adhesive - membrane - triple filter paper - sponge - positive electrode.
6. Transfer the membrane, usually for 2 hours. 7.
7. After transferring, the membrane is stained with 1×Lichun red dye solution for 5 min (shaking on decolorizing shaker). Then rinse off the unstained dye with water to see the protein on the membrane. Dry the membrane and set aside.
8. Antigen Detection
Sealing of non-specific protein binding sites on the immunoblotting membrane:
a. The transferred membrane is first moistened and washed twice with TBST for five minutes each time. Low speed horizontal shaker, room temperature.
b. Seal with 5% skim milk (TBST preparation) for one to two hours at room temperature. The containment process should be done at room temperature on a horizontal shaker at low speed.
Antibody-antigen specific binding: primary antibody incubation
9-11. Wash the membrane with TBST three times for ten minutes each time. 12.
12. Incubate with secondary antibody for one to two hours at room temperature. 13-15.
13-15. Wash the membrane with TBST three times for ten minutes each. 16.
16. Prepare ECL substrate, 2 ml (1 ml A plus 1 ml B) is sufficient for an 8 x 5 cm membrane. Moisten the membrane with the substrate for 1 minute, then remove and blot excess substrate from the membrane with filter paper. 17.
17. Expose the film by x-ray, adjust the exposure time according to the strength of the signal, generally 1 min or 5 min, or choose different times to press the film several times to achieve the best effect; after the exposure is completed, open the X-ray film clip, take out the X-ray film, and quickly immerse it in the developer to develop the film, and then terminate the development when there are obvious bands.
Western blot Frequently Asked Questions Analysis Guide
Why are the bands in electrophoresis thick?
Thick bands in electrophoresis are common, mainly due to the lack of concentration.
Treatment: Increase the length of the gel concentrate appropriately; make sure the pH of the gel concentrate reservoir is correct; reduce the electrophoretic voltage appropriately;
Why the electrophoresis voltage is high while the current is low?
This phenomenon is generally easy for beginners to appear. For example, the voltage is above 50v, but the current is below 5mA. It is mainly because the electrophoresis tank is not assembled correctly and the current does not form a path. Including: a. Inside and outside tanks are installed in reverse; b. Outside tank liquid is too little; c. Insulator at the bottom of electrophoresis tank is not removed (e.g. rubber skin for pouring glue).
Treatment: The electrophoresis tank can be assembled correctly.
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