Far Western Protocol

1. Run samples out on a gel. For bacterially expressed proteins, generally 5 μl is plenty (1ml cell cμlture; cells resuspeded in 50 μl loading buffer). Run the gels (BioRad mini gels) at 195 V for approximately 40 min. (until samples run close to the bottom of the gel).

2. Transfer the proteins from the gel onto nitrocellμlose. For the BioRad setup, the case shoμld be set up as follows: black side down, then 3M Scotch Brite Pad, then blotting paper, gel, nitrocellμlose, 2nd blotting paper, 2nd Scotch Brite Pad, and the clear side of the case. Put the case in the holder, black side of the case facing black side of the holder. Run the transfer at 100V for 1 hour.

3. Meanwhile, prepare 500 ml of AC Buffer (+ Tween):

50 ml glycerol (= 10% glycerol)

10 ml 5M NaCl (= 100mM NaCl)

10 ml1M Tris, pH 7.6 (= 20 mM Tris)

1 ml 0.5M EDTA (= 0.5mM EDTA)

5 ml 10% Tween-20 (= 0.1% Tween-20)

put on ice

b) Make 50 ml (or more) of 2% milk powder solution:

50 ml AC Buffer

1 g milk powder

-put on a rocker to dissolve the milk powder (may take 20-30 minutes). Then put on ice

4. Make the probe, using the TnT (Promega) Reticμlocyte Lysate kit.

Set up either a 25 μl reaction or a 50 μl reaction, depending on the size of tray you'll be using for washes and probing. Below is the recipe for a 50 μl reaction mix:

25 μl Reticμlocyte lysate (I use a little more, ~27 μl)

2 μl Reaction Buffer

1 μl T7 (or T3) polymerase (or other polymerase)

1 μl amino acids minus methionine (or missing other amino acid)

1 μl RNA Guard

4 μl 35S-met (or other labelled amino acid)

16 μl DNA + ddH2O (1-2 μg DNA)

Spin down the sample (pμlse spin) to remove air bubbles. Let the reaction proceed at room temperature for 1 hour, 10 minutes (can be longer or shorter, depending on the protein).

Block, Probe, and Wash

5. After the transfer is complete, put the blot into a tray. Keep the side that was touching the gel up). Do 1-2 quick washes with 1X PBS to remove the SDS. Then, do a quick rinse in AC Buffer, to remove the PBS. Pour on the 2% milk powder (just enough to cover the blot), put a lid on the dish, and rock the blot at 4℃ for 1 hour. This is the blocking step.

6. Meanwhile, set up 2 spin columns:

Use 1cc syringes, remove the cap and the plunger. Put in glass wool to fill the opening at the bottom, push down with the plunger. Put the syringe in a 15 mL falcon tube. Add BioRad G 25 resin, stored at 4℃ in TE buffer, to the syringe , filling to the top (avoid air bubbles!). Spin down at 2000 rpm for 3 minutes. Then refill with G-25, and repeat the spin cycle. (At this point, the G-25 shoμld be packed down such that it occupies about 0.8ml.

Add to the columns 100 μl AC Buffer, spin at 2000 rpm for 3 min. Repeat two more times.

7. When the probe labeling reaction is finished, dilute the sample using AC Buffer to make a final volume of 100 μl. Load this on the spin column and spin at 2000 rpm for 3 minutes in a fresh falcon tube. Collect the flow-through. This process removes unincorporated nucleotides.

8. When there are approximately 20 minutes left in the blocking step, it is time to prepare the probe mix. At this point, you may want to collect a 2μl sample of your probe (from the 100 μl), and combine it with 10 μl 1X SDS gel loading buffer. You can then run this sample on a gel, and put film on the dried gel to see if the probe labelling step actually worked.

The volume of the probe mix depends on your initial probe reaction mix volume, and the size of tray that you are using. For example, if you set up a 50 μl initial reaction, you can use up to 20 ml of probe mix. For a 25 μl reaction, use no more than 10 ml of probe mix.