분자생물학실험
SUBJECT
Western blotting
분자생물학실험
이번 주
다음 주
분자생물학실험
RuBisCo (Ribulose-1,5-bisphosphate carboxylase oxygenase)
분자생물학실험
Materials & Method
Protein extraction (sample preparation)
1. Harvest tissue samples in liquid N2.
2. Total plant proteins were extracted by grinding in 200ul extraction buffer.
Reagent
Volume
Final conc.
1M Tris, pH7.5
1ml
100mM
sucrose
1g
10%
0.5M EDTA
0.1ml
5mM
D.W
Total
10ml
3. Centrifuge 5min at RT
4. Transfer supernatant fractions to new tubes
5. Measure a protein concentration by Bradford assay.
생략
분자생물학실험
Materials & Method
Electrophoresis
1. Mix extract with 4x sample buffer and boiling 5min. (each samples up to 10~20ug)
2. Load the samples. (sample: 15ul, marker: 5ul)
3. Run on an SDS-PAGE mini-gel until the blue front is at the bottom of the gel.
┗(100v ->150V for 1hr)
분자생물학실험
Materials & Method
Transfer to membrane
1. Cut off stacking gel
2. Measure the dimensions of the gel and note the positions of the ladder bands.
3. Agitate the gel in TGM for 15-20min at RT
4. Prepare the transfer stack as follows.
5. Put the tank in box containing ice.
6. 100V 0.5A for 1hr
분자생물학실험
분자생물학실험
Materials & Method
Immunodetection
1.
Block non-specific binding sites by immersing the membrane in 5% non-fat dried milk (ECL
Blocking Agent – RPN2125) in PBST of TBST for 1hr at RT.
2. Wash 2X briefly with PBST and 2X for 5min.
3. Incubate the membrane in PBST with 1/1000 Primary antibody for 1hr at RT on shaker.
4. Wash 2X briefly with PBST, once for 15min and 3X for 5min.
5. Incubate the membrane in PBST with 1/10000 secondary antibody for 1hr at RT.
6. Wash 2X briefly with PBST, once for 15min and 3X for 5min.
7. Wash with PBS for 5min to remove Tween-20.
8. Mix ECL-Plus detection solution (GE Healthcare RPN2132) A and B in a ratio of 40:1 (A:B =
2ml:50ul) The final volume of detection reagent required is 0.1 ml/cm2)
9. Place membrane on SaranWrap and drop the detection reagent on to the membrane.
10. Incubate for 5min RT at dark.
11. Chemiluminescent detection.
분자생물학실험
GFP (Green Fluorescent Protein)
분자생물학실험
proSHR
SHR
GFP
proSCR
GFP
SCR
분자생물학실험
BUFFER
PROTEIN extraction buffer
4x SDS-PAGE sample buffer (loading dye처럼 사용)
Reagent
Volume
Final conc.
1M Tris, pH7.5
1ml
100mM
sucrose
1g
10%
0.5M EDTA
0.1ml
5mM
D.W
Reagent
Volume
Final conc.
1M Tris/HCl (pH6.8)
2ml
200mM
Glycerol
4ml
40%
10% SDS
8ml
8%
1% Bromophenolblue
0. ml
0.05%
D.W.
Up to
Total
10ml
10ml
Total
10X running buffer
(SDS-PAGE 젤 내릴 때 사용)
1X Transfer buffer(TGM)
Transfer할 때 사용
Adding 10mM DTT
10X TG salt
Reagent
Volume
Final conc.
Tris/HCl
30.2g
250mM
Reagent
Volume
Reagent
Volume
Final conc.
Glycin
150g
2M
10X TG salt
100ml
Tris/HCl
30.2g
250mM
10% SDS
10g
1%
MeOH
200ml
Glycin
150g
2M
D.W
700ml
D.W
Total
1000ml
Total
D.W
Total
1000ml
pH8.3 use 1X buffer, do not use again.
1000ml
No need to pH.
0.02% SDS (1ml 20% SDS/L) – optional
1X Phosphate-buffered saline (PBS)
immunodetection시 사용하게 될 buffer
Reagent
1X Volume
10X Volume
Final conc.
NaCl
8g
80g
137mM
KCl
0.2g
2g
2.7mM
Na2HPO4
1.44g
14.4g
10mM
KH2PO4
0.24g
2.4g
2mM
1000ml
1000ml
D.W.
Total
PBST (1X PBS with 0.1% Tween-20)
분자생물학실험