Antibody Clonotype Analysis Using Affinity Immunoblotting Protocol

Materials of Affinity Immunoblotting Protocol

Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ-cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials. We do not add sodium azide to reagents.

1. 25 % glycerol (v/v): Add 25 mL glycerol to 75 mL of distilled water. Mix well.
2. 5Xacrylamide (26.5 % T, 3 % C): Add about 25 mL water to a 100 mL graduated cylinder or a glass beaker. Weigh 12.84 g acrylamide and 0.4098 g bis acrylamide and transfer to the cylinder. Add a spatula of AG 501-X8 (D) mixedresin beads and stir using a magnetic stir bar on a magnetic plate for about 30 min. Make up to 50 mL (after removing the stir bar) with water and filter through a 0.45μm Corning filter. Store at 4 °C, with bottle wrapped with aluminum foil.
3. 10 % Tween-20: Add 90 mL of distilled water into a glass beaker. Add 10 mL Tween-20 and mix.
4. 2 % ammonium persulfate: Weigh 0.02 g ammonium persulfate and dissolve in 1 mL of distilled water.
5. N, N, N', N'-Tetramethyl-ethylenediamine. Store at 4 °C.
6. Alkaline phosphatase buffer: Weigh 6.1 g of Tris, 2.9 g sodium chloride, and 0.51 g magnesium chloride-6H₂O and make it to 500 mL with water after adjusting pH to 9.3 with HCl. Store at 4 °C.
7. Nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP): Dissolve 1 g NBT in 20 mL of 70 % dimethylformamide (DMF). Dissolve 1 g BCIP in 20 mL of 100 % DMF. Add 33μL of BCIP and 66μL of NBT to 10 mL of alkaline phosphatase buffer just before adding to membrane. Alternatively, use 1-Step™ NBT/BCIP readymade mix.
8. Nitrocellulose membrane.
9. 0.5 M sodium bicarbonate solution, pH 9.5.
10. Phosphate buffered saline (PBS), pH 7.4.
11. PBS containing 0.05 % Tween-20 (PBST).
12. Ampholytes: pH 3-10 and pH 8-10.5.
13. Ro 60 autoantigen.
14. Glass plates: Two 5″ by 4″ glass plates.
15. Medium binder clips (1¼ in.).
16. Small binder clips (¾ in.).
17. Gasket with three edges, about 3 mm wide, to serve as spacer between the plates.
18. LKB-2117 Multiphor apparatus for IEF.
19. Model 3000/300 power supply.
20. pH 3 and pH 10 solutions.
21. Helium gas.
22. Sample applicator strip.
23. Paper wicks.

Methods of Affinity Immunoblotting Protocol

All procedures are at room temperature unless otherwise specified.

1. The night before focusing cut a piece of nitrocellulose membrane according to the size that would fit a small pipet box lid (yellow tip box). Add antigen (Ro 60) at 10 μg/mL of sodium bicarbonate, pH 9.5, and incubate this with the membrane overnight with shaking.
2. Pipet 5.6 mL of distilled water into a conical flask. Add 2 mL of 25 % glycerol followed by 2.1 mL of the 5×acrylamide solution. Then add 300μL of pH 3–10 ampholytes followed by 100μL of pH 8–10.5 ampholytes.
3. Degas this solution by bubbling helium through it for 15 min. Rinse the metal end of degassing tube first with water and wipe dry with Kimwipes.
4. While the solution is degassing, set up the gel apparatus. Soak the gasket in water for few minutes. Mop dry with Kimwipes.
5. Take one glass plate and lay the gasket on top of the glass plate around the edges so that it will seal the bottom and two sides of the plates. Lay the other glass plate on top of the gasket. Clamp the clips around the edges of the plates (bottom, the left side, and the right side). Stand the gel upright on using the base of the clips to pour the gel. Prepare a 2 % ammonium persulfate solution fresh.
6. After degassing is complete, the metal end of the degassing tube is cleaned with water. To the degassed solution, add 100μL of 10 % Tween-20 and mix gently. Then add 100μL of 2 % APS. Have a Pasteur pipette ready for pouring the gel. Add 10 μL of TEMED and mix gently. Pipet the gel mixture into the Pasteur pipette and transfer into the gel apparatus quickly. Attempts should be made to avoid bubbles. Fill up the gel apparatus to the top. Polymerization should begin within minutes. However, let the assembly stand for 2 h without disturbance.
7. Turn cooling unit on and set it on 4 °C in preparation for focusing.
8. After 2 h carefully remove one of the glass plates and gasket. The gel will remain on one of the glass plates.
9. Lay the glass plate on top of the IEF unit, with the gel side facing up (wipe of water on top of the unit beforehand). Place the smaller cover in place and press down slightly so as to make imprints for the wicks. Cut two wicks to the size of the gel (be as close as possible). Soak the top wick in Serva pH 3 solution and the bottom wick in Serva pH 10 solution. Dab off excess solution and place where imprints were made by cover.
10. Put smaller cover back on, making sure connection is made with both wicks. Connect red and black wires. Put on larger cover and make connections to power supply (red = +ve ; black = −ve ).
11. Prefocus by setting constant voltage 200 V for 20 min, then increase voltage to 400 V for another 20 min. Prepare samples for application.
12. Turn of power supply, disconnect wires, and remove covers. Take applicator strip and lay on top of gel 1–2 cm below top wick. Make sure strip is stuck to the gel well. Strip can hang over gel a little. Apply the samples, being careful not to spill over into other wells. Replace the covers and make the connections. Turn the power supply on to 12 W constant power. Focus for approximately 1–2 h. When focusing, the voltage will rise, and the current will drop. The rate at which these two parameters change is much faster in the beginning than the end. The run is complete when the voltage is between 1,800 and 2,000 V and the current 3–5 A. When the change appears to be very slow or not at all, turn off the unit.
13. Thirty minutes prior to end of the run, rinse nitrocellulose membrane three times with PBS, pH 7.4, shake with PBST for 30 min, and rinse three times with PBS.
14. After the run is complete, transfer the focused protein from the gel to membrane. Take gel off the flatbed and remove applicator strip. Place nitrocellulose membrane between two Kimwipes and dab dry. Place the membrane over bottom half of gel above the bottom wick. The membrane may cover the applicator strip area.
15. Place gel in a Tupperware container with a moist towel, cover and place in an oven at 37 °C for 20 min.
16. Take membrane off gel. Rinse with 200 mL of deionized water two to three times.
17. Rinse three times with PBS. Wash for 20–30 min in PBST and rinse three times with PBS.
18. Add appropriate alkaline phosphatase conjugate (10 mL) to membrane and shake for 1 h.
19. Develop bands with NBT/BCIP.
20. Scan the membrane and save results.