WESTERN PROTOCOL
互联网
WESTERN PROTOCOL |
A. Preparation of cell lysates
- Collect cells (confluent T-25) by trypsinization and spin.
-
Lyse the pellet with 100 µl lysis buffer on ice for 10 min.
For 500,000 cells, lyse with 20 µl. - Spin at 14,000 rpm (16,000 g) in an Eppendorf microfuge for 10 min at 4°C.
- Transfer the supernatant to a new tube and discard the pellet.
-
Determine the protein concentration (Bradford assay, A280, or BCA)
(We use the Bradford assay from Bio-Rad.) - Take x µl (= y µg protein) and mix with x µl of 2x sample buffer.
- Boil for 5 min.
- Cool at RT for 5 min.
- Flash spin to bring down condensation prior to loading gel.
B. Polyacrylamide gel (14.5 cm x 16.5 cm)
-
Agarose plug:
1% agarose dissolved in 1x Resolving gel buffer.
(I make 50 ml, keep melting it as I need it, and re-adding water to maintain agarose conc.) -
Resolving gel: 24 ml of a 9% gel
5.4 ml 40% acrylamide/bisacrylamide (29:1 mix)
3 ml 8x Resolving gel buffer
15.6 ml water
12 µl TEMED
60 µl 20% ammonium persulfate -
Stacking gel: 8 ml
1 ml 40% acrylamide/bisacrylamide (29:1 mix)
2 ml 4x Stacking gel buffer
5 ml water
8 µl TEMED
21.6 µl 20% ammonium persulfate
C. Preparation of gel
- Assemble the glass plates and spacers (1.5 mm thick).
- Pour an agarose plug (1-2 mm).
- Pour the running gel to about 1 cm below the wells of the comb (~20 ml).
-
Seal with 1 ml water-saturated 1-butanol.
(Can stop here and leave gel as is overnight if you want.) - When gel has set, pour off the butanol and rinse with deionized water.
- Pour the stacking gel (~5 ml) and insert the comb immediately.
- When the stacking gel has set, place in gel rig and immerse in buffer.
- Prior to running the gel, flush the wells out thoroughly with running buffer.
D. Running the gel
- After flash spinning the samples, load into the wells.
-
Be sure to use markers.
We use 15 µl Bio-Rad Kaleidoscope Prestained Standards #161-0324 directly. - Run with constant current (35 - 37 mA with voltage set at > 300 V).
- Usual running time is about 2.5 hr.
E. Using precast gels (Ready Gels from Bio-Rad):
- Assemble gel in gel rig.
- Prepare protein samples (10 µg will suffice).
- Use 5 µl of Kaleidoscope standard.
- Run at 200 V (constant voltage) for 30 min.
F. Preparation of membrane
- Cut a piece of PVDF membrane (Millipore Immobion-P #IPVH 000 10).
- Wet for about 30 min in methanol on a rocker at room temp.
- Remove methanol and add 1x Blotting buffer until ready to use.
G. Membrane transfer
- Assemble "sandwich" for Bio-Rad's Transblot.
-
Prewet the sponges, filter papers (slightly bigger than gel) in 1x Blotting buffer.
Sponge - filter paper - gel - membrane - filter paper - sponge -
Transfer for 1 hr at 1 amp at 4°C on a stir plate.
Bigger proteins might take longer to transfer.
For the Mini-Transblot, it's 100 V for 1 hr with the cold pack and prechilled buffer. - When finished, immerse membrane in Blocking buffer and block overnight.
H. Antibodies and detection
- Incubate with primary antibody diluted in Blocking buffer for 60 min at room temp.
- Wash 3 x 10 min with 0.05% Tween 20 in PBS.
- Incubate with secondary antibody diluted in Blocking buffer for 45 min at room temp.
- Wash 3 x 10 min with 0.05% Tween 20 in PBS.
- Detect with Amersham ECL kit (RPN 2106).
I. Stripping blot
- Rinse blot off with 0.05% Tween 20 in PBS.
- Put blot into Kapak bag cut to slightly bigger size than blot.
- Add about 5 to 10 ml Stripping buffer.
- Remove as much air as possible and seal bag.
- Immerse into 80°C water bath and incubate for 20 min.
- Rinse blot off with 0.05% Tween 20 in PBS.
- Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween 20.
Buffers for Westerns
- Lysis buffer:
- 0.15 M NaCl
- 5 mM EDTA, pH 8
- 1% Triton X100
- 10 mM Tris-Cl, pH 7.4
- Just before using add: 1:1000 5 M DTT
- 1:1000 100 mM PMSF in isopropanol
-
1:1000 5 M e- aminocaproic acid
- 2x sample buffer:
- 130 mM Tris-Cl, pH8.0
- 20% (v/v) Glycerol
- 4.6% (w/v) SDS
- 0.02% Bromophenol blue
-
2% DTT
- 8x Resolving gel buffer: 100 ml
- 0.8 g SDS (add last)
- 36.3 g Trizma base (= 3 M)
-
Adjust pH to 8.8 with concentrated HCl
- 4x Stacking gel buffer: 100 ml
- 0.4 g SDS (add last)
- 6.05 g Trizma base (= 0.5 M)
-
Adjust pH to 6.8
- 10x Running buffer: 1 L
- 30.3 g Trizma base (= 0.25 M)
- 144 g Glycine (= 1.92 M)
- 10 g SDS (= 1%)--add last
-
Do not adjust the pH!!
- 10x Blotting buffer: 1 L
- 30.3 g Trizma base (= 0.25 M)
- 144 g Glycine (= 1.92 M)
-
pH should be 8.3; do not adjust
- To make 2 L of 1x Blotting buffer:
- 400 ml Methanol
- 200 ml 10x Blotting buffer
-
1400 ml water
- Blocking buffer: 0.5 L
- 3% Bovine serum albumin (Fraction V)
- Make up in PBS and sterile filter.
- Then add 0.05% Tween 20.
-
Keep at 4°C to prevent bacterial contamination.
- Stripping buffer: 0.5 L (sterile filter solution and keep at 4°C)
- 0.2 M Glycine, pH 2.5
- 0.05% Tween 20