1. Run samples out on a gel. For bacterially expressed proteins, generally 5 uL is plenty (1ml cell culture; cells resuspeded in 50 ul 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 nitrocellulose. For the BioRad setup, the case should be set up as follows: black side down, then 3M Scotch Brite Pad, then blotting paper, gel, nitrocellulose, 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 mL 1M 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) Reticulocyte Lysate kit.
Set up either a 25 uL reaction or a 50 uL reaction, depending on the size of tray you'll be using for washes and probing. Below is the recipe for a 50 uL reaction mix:
25 uL Reticulocyte lysate (I use a little more, ~27 uL)
2 uL Reaction Buffer
1 uL T7 (or T3) polymerase (or other polymerase)
1 uL amino acids minus methionine (or missing other amino acid)
1 uL RNA Guard
4 uL 35S-met (or other labelled amino acid)
16 uL DNA + ddH2O (1-2 ug DNA)
Spin down the sample (pulse 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 C 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 C 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 should be packed down such that it occupies about 0.8ml.
Add to the columns 100 uL 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 uL. 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 2uL sample of your probe (from the 100 ul), and combine it with 10 uL 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 uL initial reaction, you can use up to 20 mL of probe mix. For a 25 uL reaction, use no more than 10 ml of probe mix.
|
SMALL TRAY |
LARGE TRAY |
| 10 mL 2% milk powder sltn. | 20mL 2% milk powder sltn. |
| 10 uL 1M DTT | 20 uL 1M DTT |
| 25 uL probe rxn. | 50 uL probe rxn. |
Let the probe incubate in the mix for the remaining 20 minutes, on ice.
9. When the blocking step is over, pour off the milk powder solution, and pour on the probe mix. Cover the tray. Rock the blot in the cold room for 2-3 hours.
10. When you are finished probing, pour the probe mix into a suitable radioactive waste container. Then do 2 quick washes with AC Buffer. Wash with remaining 2% milk powder solution (+ DTT), and let the blot rock in the cold room for 15 min. (make sure you have enough solution to cover the blot completely).
11. Do a quick wash in AC Buffer, and follow with 4 x 20 min washes in AC buffer, in the cold room. (use at least enough to cover the blot)
12. Let the blot dry on blotting paper. After 30-40 minutes, transfer to a new piece of blotting paper. Add baby powder to prevent sticking. Spread GENTLY. Make sure the side of the membrane that was in contact with the gel is the side that is up. Put on x-ray film, and expose.
note:
-if preferred, the blocking step can go overnight (the step before probing), rather than for an hour. So, if you are pressed for time, this is one point where you can spread the Far Western over two days.
-Denaturation/Renaturation steps can also be tried to optomize signal: noise. For example, right after the transfer, the blot is washed with 6M Guanidine-HCl in AC Buffer plus 2% milk powder (and DTT) for 30 minutes at room temperature (use 8M stock). Then it is washed in 3M Guanidine-HCl in 2% milk powder (in AC Buffer) for 30 minutes at RT; then 1M Guanidine-HCl in 2% milk powder (in AC Buffer) for 30 minutes at 4 C, and then in 0.1M for 30 minutes at 4 C. Finally, it is put in 2% milk powder solution, in AC Buffer, with DTT, overnight at 4 C. Proceed with probing the next morning.
Below is a table showing how to make the Guanidine-HCl solutions.
|
[guanidine-HCl] |
6M |
3M |
1M |
0.1M |
2% M.P. |
| glycerol | 2.5 mL | 2.5 mL | 2.5 mL | 2.5 mL | 2.5 mL |
| 5M NaCl | 0.5 mL | 0.5 mL | 0.5 mL | 0.5 mL | 0.5 mL |
| 1M Tris, PH 7.5 | 0.5 mL | 0.5 mL | 0.5 mL | 0.5 mL | 0.5 mL |
| 0.5M EDTA | 0.05 mL | 0.05 mL | 0.05 mL | 0.05 mL | 0.05 mL |
| 10% Tween-20 | 0.25 mL | 0.25 mL | 0.25 mL | 0.25 mL | 0.25 mL |
| 8M Guanidine-HCl | 18.75 mL | 9.30 mL | 3.13 mL | 0.31 mL | ------- |
| Milk Powder | 0.5 g | 0.5 g | 0.5 g | 0.5 g | 0.5 g |
| 1M DTT | 25 uL | 25 uL | 25 uL | 25 uL | 25 uL |
| ddH2O | 2.45 mL | 12.82 mL | 18.07 mL | 20.89 mL | 21.20 mL |
| Total Volume | 25 mL | 25 mL | 25 mL | 25 mL | 25 mL |
Far Western blot of FTZ polypeptides expressed in bacteria and detected by 35S FTZ-F1
