1. Western blotting 분자생물학실험 SUBJECT

2. 분자생물학실험 이번 주 다음 주

3. 분자생물학실험 RuBisCo (Ribulose-1,5-bisphosphate carboxylase oxygenase)

4. 분자생물학실험 Materials & Method ReagentVolumeFinal conc. 1M Tris, pH7.5 1ml100mM sucrose 1g10% 0.5M EDTA 0.1ml5mM D.W Total 10ml 1.Harvest tissue samples in liquid N2. 2.Total plant proteins were extracted by grinding in 200ul extraction buffer. 3. Centrifugation 5min at RT 4. Transfer supernatant (150ul) fractions to new tubes 5. Measure a protein concentration by Bradford assay. Protein extraction (sample preparation) 생략

5. 분자생물학실험 Materials & Method 1.Mix extract with 4x SDS-PAGE sample buffer ( with DTT) and boiling 5min. 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) Electrophoresis

6. 분자생물학실험 Materials & Method 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, agitate the membrane in Methanol 4. Prepare the transfer stack as follows. Transfer to membrane 5. Put the tank in box containing ice. 6. 100V 0.5A for 1hr

7. 분자생물학실험

8. Materials & Method 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. Immunodetection

9. 분자생물학실험 GFP (Green Fluorescent Protein)

10. 분자생물학실험 SHR GFP proSHR SCR GFP proSCR

11. 분자생물학실험 BUFFER PROTEIN extraction buffer ReagentVolumeFinal conc. 1M Tris, pH7.5 1ml100mM sucrose 1g10% 0.5M EDTA 0.1ml5mM D.W Total 10ml 4x SDS-PAGE sample buffer (loading dye 처럼 사용 ) ReagentVolumeFinal conc. 1M Tris/HCl (pH6.8)2ml200mM Glycerol4ml40% 10% SDS8ml8% 1% Bromophenolblue0. ml0.05% D.W.Up to Total10ml Adding 10mM DTT 10X running buffer (SDS-PAGE 젤 내릴 때 사용 ) ReagentVolumeFinal conc. Tris/HCl 30.2g250mM Glycin 150g2M 10% SDS 10g1% D.W Total 1000ml pH8.3 use 1X buffer, do not use again. 1X Transfer buffer(TGM) Transfer 할 때 사용 ReagentVolume 10X TG salt100ml MeOH200ml D.W700ml Total1000ml 10X TG salt ReagentVolumeFinal conc. Tris/HCl30.2g250mM Glycin150g2M D.W Total1000ml No need to pH. 0.02% SDS (1ml 20% SDS/L) – optional

12. Reagent1X Volume10X VolumeFinal conc. NaCl8g80g137mM KCl0.2g2g2.7mM Na2HPO41.44g14.4g10mM KH2PO40.24g2.4g2mM D.W. Total1000ml 1X Phosphate-buffered saline (PBS) immunodetection 시 사용하게 될 buffer PBST (1X PBS with 0.1% Tween-20) 각각의 step 에 사용되는 buffer 들의 조성을 표로 정리 해 봤어요 Volume 이나 concentration 은 신경 쓰지 않아도 됩니다. 이 시약들의 간단한 원리 및 역할을 작성해 주세요.

13. 분자생물학실험