Exercise 4.8 - Western Blots

LEVEL III

Figure 4.15 Samples of a Western blot

Materials

• Blot Cell
• BA 83 0.2 µ m pore nitrocellulose sheets
• Buffer, PBS-Tween 20
• Antigenic proteins, antibodies, and horseradish peroxidase labeled antiglobulins

Procedure 21

1. Run an electrophoretic separation of known antigenic proteins according to the procedures in Exercise 4.1 and 4.2.

2. Draw a line 0.5 cm from the top edge of an 8 x 10 cm nitrocellulose sheet and soak it in blot buffer for about 5 minutes.

   Nitrocellulose is both fragile and flammable and easily contaminated during handling. Wear gloves which are prewashed.

   When soaking the nicrocellulose wet first one side and then turn the sheet over and wet the other, to prevent trapping air within the filter.

3. Place 200 ml of blot buffer into a tray and add a piece of filter paper slightly larger than the electrophoretic gel from Step 1.

4. Remove the gel from the electrophoresis chamber after the proteins have been separated, and place the gel into the tray containing the filter paper. Do not allow the gel to fall onto the paper, but place it next to the paper in the tray.

5. Gently slide the gel onto the top of the filter paper. Keep the stacking gel off of the paper until the last moment, since it tends to stick and make repositioning difficult.

6. Holding the gel and the filter paper together, carefully remove them from the tray of blot buffer and transfer the paper and gel to a pad of the blot cell with the gel facing up.

7. Transfer the nitrocellulose sheet (ink side down) onto the top of the gel and line up the line drawn on the sheet with the top of the stacking gel.

   Once the gel and nitrocellulose touch they can not be separates.

8. Roll a glass rod across the surface of the nitrocellulose to remove any air bubbles and insure good contact between the gel and nitrocellulose.

9. Lay another sheet of wet filter paper on top of the nitrocellulose creating a sandwich of paper-gel- nitrocellulose-paper, all lying on the pad of the blot cell.

10. Add a second pad to the top of the sandwich and place the entire group inside of the support frame of the blot cell, and assemble the blot cell so that the nitrocellulose side of the sandwich is toward the positive terminal.

11. Check that the buffer levels are adequate and that the cooling water bath is adjusted to at least 5° C. Subject the gel to electrophoresis for 30 minutes with the electrodes in the high field-intensity position. Follow the manufacturer directions during this phase. Failure to closely monitor the electrophoresis buffer or temperature can result in a fire. Use a circulating cold bath appropriate to the apparatus and hold the voltage to a constant 100 vdc.

12. Upon completion of the electrophoresis (timed according to manufacturer's directions), turn off the power and disassemble the apparatus. Remove the blot pads from the sandwich and remove the filter paper from the nitrocellulose side.

13. Place the sandwich, nitrocellulose side down, onto a glass plate and remove the other filter paper.

14. Use a ball point pen to outline the edges of the separating gel onto the nitrocellulose, including the location of the wells. Carefully lift the gel away from the nitrocellulose and mark the locations of the pre-stained molecular weight standards as the gel is peeled away. Peel the gel from the separating gel side, not the stacking gel.

15. Wash the blot (the nitrocellulose sheet) at least four times with 100 ml of PBS-Tween 20 for five minutes each on a rocking platform.

16. Cut the blot into 0.5 cm strips.

17. Inactivate sera containing positive and negative antibody controls to the antigens under examination by treating at 56 ° C for 30 minutes. Make dilutions of 1:100 and 1:1000 of the controls with PBS-Tween 20.

18. Place 3 ml of the diluted sera or controls onto a strip from Step 16 and incubate for 1 hour at room temperature while continuously rocking the sample.

19. Wash the strips four times for 5 minutes each with 10 ml quantities of PBS-Tween 20. The first wash should be done at 50° C but the last three may be done at room temperature.

20. Add 3 ml of horseradish peroxidase-labeled antiglobulin, optimally diluted in PBS-Tween and incubate at room temperature for 1 hour with continuous agitation.

21. Wash the strips four times for 5 minutes each with PBS- Tween 20 and one more time with PBS only.

22. Remove the PBS and add 5 ml of substrate solution. Positive reaction bands usually appear within 10 minutes. Stop the reaction by washing with water. Refer to Figure 4.15 for a comparison.

Notes

One of the more difficult tasks of electrophoretic separations is the identification of specific bands or spots within a developed gel. As observed with LDH isozymes, one method of doing this is to react the bands with an enzyme substrate that can be detected colorimetrically.

As a rule, however, most peptides are denatured during electrophoresis, and, of course, nucleic acids have no enzyme activity. The methods employed for identifying non- enzymatic proteins and nucleic acids have been termed Western for immunoblotting of proteins, Southern for techniques using DNA probes Northern when using RNA probes. The probes are radioactive complimentary strands of nucleic acid. The first of these techniques was the Southern, named for the developer of the procedure, Edward Southern. Northern and then Western blots were named by analogy.

Blotting techniques first develop a primary gel: protein on acrylamide; or DNA/RNA on agarose. The gel patterns are then transferred to nitrocellulose membrane filters and immobilized within the nitrocellulose membrane. This process of transfer to an immobilizing substrate is where the term blotting originated. The process is widely used in today's laboratories because the immobilization allows for extensive biochemical and immunological binding assays that range from simple chemical composition to affinity purification of monospecific antibodies and cell- protein ligand interactions.

In practice, the electrophoresis gel is sandwhiched between two layers of filters, two foam pads (for support) and two layers of a stainless steel mesh. This entire apparatus can be submerged in a buffer and transfer allowed to occur by diffusion (yielding two blots, one on each filter), or can be arranged in an electro-convective system so that transfer occurs in a second electrophoretic field.

Once the transfer has occurred, the blots can be probed with any number of specific or non-specific entities. DNA can be probed, for example, with cDNA or even a specific messenger RNA to identify the presence of the gene for that message.

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