原创：western protocol
丁香园论坛
1792
做了两个月的时间的 western blotting 终于出了比较好的结果。
老板让整理出protocol
这是写的PROTOCOL
大概会有很多错误请大家指正！谢谢先
WESTERN PROTOCOL
A. Preparation of Samples
1. Get the heads of the fly
2. Stir them in the lysis buffer(4heads/10ul)
3. Add 2XSDS-gel-loading buffer(4heads/10ul)
4. Spin at 112000 rpm in an Eppendorf microfuge for 10 min at 4°C.
5. Transfer the supernatant to a new tube and discard the pellet.
6. Boil for 3 min at 99°C.
7. store at 4°C.
B. Polyacrylamide gel
1. Resolving gel: 10 ml of a 10% gel
4 ml H2O
3.3ml 30% acrylamide/bisacrylamide (29:1 mix)
2.5 ml1.5M Tris(PH 8.8)
10% SDS 100 µl
10% ammonium persulfate 100 µl
TEMED 8 µl
2. Stacking gel: 4 ml
2.7ml water 1 ml
0.67ml 30% acrylamide/bisacrylamide (29:1 mix)
0.ml 1.0M Tris(PH=6.8)
10% SDS 40 µl
10% ammonium persulfate 40 µl
TEMED 7 µl
C. Preparation of gel
1. Assemble the glass plates and spacers (0.75 mm thick).
2. Pour the running gel to about 1 cm below the wells of the comb (~3.2 ml).
3. Seal with 1 ml anhydrous ethanol
(Can stop here and leave gel as is overnight if you want.)
4. When gel has set, pour off the anhydrous ethanol and rinse with deionized water.
5. Pour the stacking gel (~1.2 ml) and insert the comb immediately.
6. When the stacking gel has set, place in gel rig and immerse in buffer.
7. Prior to running the gel, flush the wells out thoroughly with running buffer.
D. Running the gel
1. After flash spinning the samples, load into the wells.
2. Be sure to use markers.
We use 10 µl Bio-Rad Kaleidoscope Prestained Standards #161-0324 directly.
3. Run with constant voltage (120V)
4. Usual running time is about 1：40
F. Preparation of membrane
1. Cut a piece of PVDF membrane(Pall Corporation ).
2. Wet for about 30 min in methanol at room temp.
3. Remove methanol and add 1x Blotting buffer until ready to use.
G. Membrane transfer
1. Assemble "sandwich" for Bio-Rad's Transblot.
2. Prewet the sponges, filter papers (slightly bigger than gel) in 1x Blotting buffer.
Sponge - filter paper - gel - membrane - filter paper - sponge
3. 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.
4. When finished, immerse membrane in Blocking buffer and block overnight.
H. Antibodies and detection
1. Incubate with primary antibody diluted in Blocking buffer for 2 hours at room temp.
2. Wash 3 x 10 min with PBST. 1 x10min secondary washing solution
3. Incubate with secondary antibody diluted in Blocking buffer for 60 min at room temp.
4. 3 x10min secondary washing solution 1 x10min alkaline phosphatese buffer
5. Detect with Chromogenic
I. Stripping blot
1. Rinse blot off with 0.05% Tween 20 in PBS.
2. Put blot into Kapak bag cut to slightly bigger size than blot.
3. Add about 5 to 10 ml Stripping buffer.
4. Remove as much air as possible and seal bag.
5. Immerse into 80°C water bath and incubate for 20 min.
6. Rinse blot off with 0.05% Tween 20 in PBS.
7. Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween 20.
Reagents and Buffers for Westerns
Reagent
30% acrylamide/bisacrylamide (29:1 mix) 100ml(dark bottle at 4°C)
29g acrlamid
1g bisacrylamid
1.5M Tris(PH 8.8) 100ml 18.171g (at4°C)
10% SDS 100 ml 10g (at room temperature)
10% ammonium persulfate 1ml 0.1g(replace every week store at at4°C)
TEMED 8 room temprature
Antibodies
Primary antibody: 5A11(anti-mouse IgG) 500x-1000x
Secondary antibody: Anti-mouse IgG-Alkaline Phospphatase
Buffers
Lysis buffer: 100ml
50 mM Tris-Cl, pH 8.0 0.6057g
150m M NaCl 0.8766g
0.02%NaN3 0.02g
100ug/mlPMSF 0.01g
10ug/ml Aprotinin 0.001g
1% Triton X100 1g
2x SDS-loading buffer: 100ml
100 mM Tris-Cl, pH8.0 1.2114
20% (v/v) Glycerol 20g
4% (w/v) SDS 4g
0.2% Bromophenol blue 0.2g
200mM DTT 20ul
5x Running buffer: 1 L
15.1 g Trizma base (= 125m M)
94g Glycine (= 1.25 M)
50ml 10%SDS (= 0.5%)--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 1L of 1x Blotting buffer:
200 ml Methanol
100 ml 10x Blotting buffer
700 ml water
Blocking buffer: 0.5 L
5%No fat milk or 3% Bovine serum albumin (Fraction V)
Make up in PBST and sterile filter.
Keep at 4°C to prevent bacterial contamination.
10xPBST 1L PH=7.4
Nacl 80g
KCl 2g
Na2HPO4 144g
KH2PO4 0.24g
Tween20 3ml
10xSecondary washing solution 1L PH=7.5
150mM NaCl 8.766g
50mM TrisCl 6.057g
Chromogenic solution
33ulNBT solution
33ulBCIP solution
10ml alkaline phosphatese buffer
NBT solution:0.1g NBT in 10ml 70% N,N-dimethylformamide
BCIP solution:0.5g BCIP in 10ml N,N-dimethylformamide
Alkaline Phosphatese buffer 1L
100mM NaCl 5.844g
5mM MgCl 1.0165g
100mM Tris.Cl(PH=9.5) 12.114g
老板让整理出protocol
这是写的PROTOCOL
大概会有很多错误请大家指正！谢谢先
WESTERN PROTOCOL
A. Preparation of Samples
1. Get the heads of the fly
2. Stir them in the lysis buffer(4heads/10ul)
3. Add 2XSDS-gel-loading buffer(4heads/10ul)
4. Spin at 112000 rpm in an Eppendorf microfuge for 10 min at 4°C.
5. Transfer the supernatant to a new tube and discard the pellet.
6. Boil for 3 min at 99°C.
7. store at 4°C.
B. Polyacrylamide gel
1. Resolving gel: 10 ml of a 10% gel
4 ml H2O
3.3ml 30% acrylamide/bisacrylamide (29:1 mix)
2.5 ml1.5M Tris(PH 8.8)
10% SDS 100 µl
10% ammonium persulfate 100 µl
TEMED 8 µl
2. Stacking gel: 4 ml
2.7ml water 1 ml
0.67ml 30% acrylamide/bisacrylamide (29:1 mix)
0.ml 1.0M Tris(PH=6.8)
10% SDS 40 µl
10% ammonium persulfate 40 µl
TEMED 7 µl
C. Preparation of gel
1. Assemble the glass plates and spacers (0.75 mm thick).
2. Pour the running gel to about 1 cm below the wells of the comb (~3.2 ml).
3. Seal with 1 ml anhydrous ethanol
(Can stop here and leave gel as is overnight if you want.)
4. When gel has set, pour off the anhydrous ethanol and rinse with deionized water.
5. Pour the stacking gel (~1.2 ml) and insert the comb immediately.
6. When the stacking gel has set, place in gel rig and immerse in buffer.
7. Prior to running the gel, flush the wells out thoroughly with running buffer.
D. Running the gel
1. After flash spinning the samples, load into the wells.
2. Be sure to use markers.
We use 10 µl Bio-Rad Kaleidoscope Prestained Standards #161-0324 directly.
3. Run with constant voltage (120V)
4. Usual running time is about 1：40
F. Preparation of membrane
1. Cut a piece of PVDF membrane(Pall Corporation ).
2. Wet for about 30 min in methanol at room temp.
3. Remove methanol and add 1x Blotting buffer until ready to use.
G. Membrane transfer
1. Assemble "sandwich" for Bio-Rad's Transblot.
2. Prewet the sponges, filter papers (slightly bigger than gel) in 1x Blotting buffer.
Sponge - filter paper - gel - membrane - filter paper - sponge
3. 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.
4. When finished, immerse membrane in Blocking buffer and block overnight.
H. Antibodies and detection
1. Incubate with primary antibody diluted in Blocking buffer for 2 hours at room temp.
2. Wash 3 x 10 min with PBST. 1 x10min secondary washing solution
3. Incubate with secondary antibody diluted in Blocking buffer for 60 min at room temp.
4. 3 x10min secondary washing solution 1 x10min alkaline phosphatese buffer
5. Detect with Chromogenic
I. Stripping blot
1. Rinse blot off with 0.05% Tween 20 in PBS.
2. Put blot into Kapak bag cut to slightly bigger size than blot.
3. Add about 5 to 10 ml Stripping buffer.
4. Remove as much air as possible and seal bag.
5. Immerse into 80°C water bath and incubate for 20 min.
6. Rinse blot off with 0.05% Tween 20 in PBS.
7. Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween 20.
Reagents and Buffers for Westerns
Reagent
30% acrylamide/bisacrylamide (29:1 mix) 100ml(dark bottle at 4°C)
29g acrlamid
1g bisacrylamid
1.5M Tris(PH 8.8) 100ml 18.171g (at4°C)
10% SDS 100 ml 10g (at room temperature)
10% ammonium persulfate 1ml 0.1g(replace every week store at at4°C)
TEMED 8 room temprature
Antibodies
Primary antibody: 5A11(anti-mouse IgG) 500x-1000x
Secondary antibody: Anti-mouse IgG-Alkaline Phospphatase
Buffers
Lysis buffer: 100ml
50 mM Tris-Cl, pH 8.0 0.6057g
150m M NaCl 0.8766g
0.02%NaN3 0.02g
100ug/mlPMSF 0.01g
10ug/ml Aprotinin 0.001g
1% Triton X100 1g
2x SDS-loading buffer: 100ml
100 mM Tris-Cl, pH8.0 1.2114
20% (v/v) Glycerol 20g
4% (w/v) SDS 4g
0.2% Bromophenol blue 0.2g
200mM DTT 20ul
5x Running buffer: 1 L
15.1 g Trizma base (= 125m M)
94g Glycine (= 1.25 M)
50ml 10%SDS (= 0.5%)--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 1L of 1x Blotting buffer:
200 ml Methanol
100 ml 10x Blotting buffer
700 ml water
Blocking buffer: 0.5 L
5%No fat milk or 3% Bovine serum albumin (Fraction V)
Make up in PBST and sterile filter.
Keep at 4°C to prevent bacterial contamination.
10xPBST 1L PH=7.4
Nacl 80g
KCl 2g
Na2HPO4 144g
KH2PO4 0.24g
Tween20 3ml
10xSecondary washing solution 1L PH=7.5
150mM NaCl 8.766g
50mM TrisCl 6.057g
Chromogenic solution
33ulNBT solution
33ulBCIP solution
10ml alkaline phosphatese buffer
NBT solution:0.1g NBT in 10ml 70% N,N-dimethylformamide
BCIP solution:0.5g BCIP in 10ml N,N-dimethylformamide
Alkaline Phosphatese buffer 1L
100mM NaCl 5.844g
5mM MgCl 1.0165g
100mM Tris.Cl(PH=9.5) 12.114g
