2) What happens when we incubate the nitrocellulose in primary antibody
The primary antibody, which is the specific antibody mentioned above, sticks to your protein and forms an antibody-protein complex with the protein of interest.
Making a primary antibodyThis description assumes you have available purified protein. Run the protein on an SDS-PAGE gel. Stain the gel with KCl. The KCl forms a precipitate with the SDS. Since the area with the protein has a low concentration of SDS, the area with the protein will not show a precipitate. This will allow you to see the protein band as a clear band against a milky white precipitate on the rest of the gel.
Carefully cut out the band and soak it in 1 mL PBS buffer. Crush it and make an emulsion with 1 mL Freund's Complete Adjuvant (which is an oily substance). The complete adjuvant contains microbacteria (an immune stimulant) to increase the immune response. Inject this subscapularly into a rabbit. This is your first inoculation. Only use the complete adjuvant for the first inoculation. NEVER inject a rabbit with complete adjuvant more than one time.
Rest the rabbit for one month, then repeat the process using an incomplete adjuvant. You can expect to see good antibody titers about 10 days after the second booster.
Bleed the rabbit. You can expect about 30 to 40 mL per bleeding, and about 50 percent of the volume is serum. Now you have rabbit antisera.
To get your primary antibody, dilute the rabbit antisera in blotto (aka Carnation Nonfat Dry Instant Milk) and apply it to your nitrocellulose blot. Make sure you dilute 1:500 to 1:100 in blotto; less dilution will give you background binding and really muddy up your results.
Secondary antibodyThis is much easier than the procedure for the primary antibody. Grab a catalogue and look for a goat-anti-rabbit antibody conjugated to horseradish peroxidase (HRP). The goat-anti-rabbit is your secondary antibody (the one that "sticks" to the primary antibody) and the HRP is the conjugated enzyme that will allow you to visualize your protein.
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