Western blotting (reagent preparation and procedure)

Western blotting (reagent preparation and procedure)

This technique involves the transfer of electrophoretically separated components from a gel to a solid support and detection using specific reagents for specific amino acid sequences as probes. The probe used in Western is an antibody that specifically reacts with an epitope presented by a target protein attached to a solid support. The role of this technique is to identify and identify certain specific proteins in a complex mixture of non-radiolabeled protein components.
Instruments : pressure cooker, electric homogenizer, high-speed centrifuge , vertical plate electrophoresis transfer device, bleaching shaker .
Reagents : single detergent lysate, separation gel, 4 % concentrated loading , G250 Coomassie Brilliant Blue solution, 5X SDS loading buffer, running buffer, transfer buffer, 10X Li Chunhong dye solution, blocking solution, TBST , TBS , developer, fixer, antibody, ECL chemiluminescence reagent.
Miscellaneous goods and consumables : various specifications of tips , centrifuge tubes and samplers; various specifications of beakers , measuring cylinders and other glass equipment; nitrocellulose membrane , latex gloves, masks, cling film, X- ray clips, X- ray film, timer, absorbent paper.

Reagent preparation:
(a) mother liquor
34.8mg/ml (200mmol/L) PMSF
PMSF 0.348g
Isopropyl alcohol 10ml
After dissolution, store at - 20 °C .

10 % SDS
SDS 5g
Distilled water to 50ml
Dissolve in a 50 ° C water bath and store at room temperature. If precipitation occurs during long-term storage, it can still be used after the water bath has melted.

5mol/L NaF sodium fluoride
Sodium fluoride 2.1g
Ultra pure water 10ml
After dissolution, store at -20 °C .

100mmol/L activated sodium citrate
Sodium metasilicate 0.122g
Ultra pure water 8ml
Concentrated hydrochloric acid to adjust the pH to 10 (color turns yellow), hot water bath at about 80 °C until the solution is colorless, cool at room temperature, reset the pH to 10 , repeat the hot water bath and adjust the pH until the solution remains colorless, constant volume 10ml , stored at -20 °C .

1.0 mol/L Tris•HCl ( pH 8.0 )
Tris (MW121.14) 1.212g
Ultra pure water 8ml
After dissolving, adjust the pH to 8.0 with concentrated hydrochloric acid , and finally make up to 10 ml with ultrapure water , and store at room temperature after high temperature sterilization.

10 % ammonium persulfate ( AP )
Persulfate amine 0.1g
Ultra pure water 1.0ml
After dissolution , store at 4 °C for 2 weeks.

1.5mol/L Tris•HCl ( pH8.8 )
Tris (MW121.14) 45.43g
Ultra pure water 200ml
After dissolving, adjust the pH to 8.8 with concentrated hydrochloric acid , and finally make up to 250 ml with ultrapure water , and store at room temperature after high temperature sterilization.

0.5mol/L Tris•HCl ( pH6.8 )
Tris (MW121.14) 0.606g
Ultra pure water 8ml
After dissolving, adjust the pH to 6.8 with concentrated hydrochloric acid , and finally make up to 10 ml with ultrapure water , and store at room temperature after high temperature sterilization.

30 % Acr/Bic ( 29 : 1 )

Acrylamide ( Acr )     14.5g
Methylene diacrylamide ( Bic ) 0.5g
Ultrapure water to         50ml
After dissolving at 37 ° C, store at 4 °C . Return to room temperature and no precipitation upon use.

20 % Tween20
Tween20 10ml
Distilled water to   50ml
Tween20 is relatively viscous. It is necessary to cut off the front end of the large gun head, expand the suction port, and slow down when sucking and pipetting. Avoid liquid hanging on the wall and store at 4 °C after mixing .

(2) using liquid

Single detergent lysate ( 50mmol/L Tris•HCl pH8.0 , 150mmol/L NaCl , 1 % SDS , 1mmol/L PMSF ):
1mol/L Tris•HCl ( pH8.0 ) 2.5ml
NaCl 0.438g
10% SDS    0.5ml
200mmol/L PMSF         2.5ml

Distilled water to 50ml

If the phosphorylated protein is detected, 100ul 5mol/L NaF and 500ul 100mmol/L activated sodium citrate should be added , mixed and stored at 4 °C .

G250 Coomassie Brilliant Blue Solution (for dyeing)
Coomassie Brilliant Blue G250 : 0.063g
Methanol:         15ml
Acetic acid:       5ml
Distilled water to      50ml
After dissolution, store at 4 °C .

Separating glue (two pieces of glue, 10ml )
        8 %                10%               12%               15%
Ultra pure water 4.6ml             4.0ml              3.3ml              2.3ml
30 % Acr/Bic ( 29 : 1 )     2.7ml              3.3ml              4.0ml              5.0ml
1.5 mol/L Tris•HCl ( pH8.8 ) 2.5ml               2.5ml              2.5ml              2.5ml
10 % SDS 100 m l             100 m l             100 m l             100 m l
10% AP (persulfate amine) 100 m l              100 m l             100 m l             100 m l
TEMED 4 m l               4 m l                4 m l                4 m l
  After adding TEMED , mix immediately to fill the gel.

4 % concentrated glue (two pieces of glue, 5ml )
Ultra pure water 2.1ml
30 % Acr/Bic ( 29 : 1 )     0.5ml
0.5 mol/L Tris•HCl ( pH6.8 ) 0.38ml
10 % SDS 30 m l  
10% AP (persulfate amine)   30 m l
TEMED   3 m l  
  After adding TEMED , mix immediately to fill the gel.

Reduced 5XSDS loading buffer ( 0.25mol/L Tris/HCl pH6.8 , 0.5mol/L dithio-tertiitol , 10 % SDS , 0.5 % bromophenol blue, 50 % glycerol)
0.5 mol/L Tris•HCl ( pH6.8 ) 0.5ml
Dithiolitol ( DTT , MW154.5 )       0.078g
SDS 0.1g
10% bromophenol blue 0.05ml
Glycerin 0.5ml

After mixing, dispense in a 1.5 ml centrifuge tube and store at 4 °C .

Electrophoresis buffer ( 25mmol/L Tris , 0.25mol/L glycine, 0.1 % SDS )
Tris ( MW121.14 )   1.515g
Glycine ( MW75.07 )     9.385g
SDS   0.5g
Distilled water to 500ml
Store at room temperature after dissolution. The secondary solution can be reused 2 to 3 times.

Transfer buffer ( 48mmol/L Tris , 39mmol/L glycine, 0.01 % SDS , 20 % methanol)
Glycine ( MW75.07 )    2.9g
Tris ( MW121.14 )      5.8g
SDS   0.1g
Methanol   200ml
Distilled water to     1000ml
Store at room temperature after dissolution. The secondary solution can be reused 2 to 3 times.

10X Li Chunhong dyeing solution
Li Chunhong S 0.2g
Trichloroacetic acid 3g
Sulfosalicylic acid 3g
Distilled water to 10ml
After dissolving, store at 4 °C in the dark , dilute it 10 times when used, and do not recycle the diluted solution.

TBS buffer ( 10mmol/L Tris/HCl pH7.5, 150mmol/L NaCl )

1 mol/ LTris•HCl ( pH7.5 ) 10ml
NaCl         8.8g
Distilled water to          1000ml

TBST buffer (TBS buffer containing 0.05% Tween20-)
20 % Tween20 1ml
TBS 400ml
It can be used after mixing, and it is best to use it now.

Blocking solution ( TBST buffer containing 5 % skim milk powder )
Skim milk powder (domestic, An Yi brand) 2.5g
TBST 50ml
Store at 4 °C after dissolution . When using, return to room temperature, use it to cover the membrane surface, and use it once.

Eluted antibody buffer ( 100mmol/L 2-Mercaptoethanol , 2 % SDS , 62.5m mol/L Tris•HCl pH6.8 )

14.4 mol/L 2- Mercaptoethanol (β- mercaptoethanol ) 700 m l   (ventilation kitchen Riga)
SDS 2g
0.5mol/L Tris•HCl ( pH6.8 ) 12.5ml
Ultrapure water to 100ml
When it is formulated, it is carried out in a ventilated kitchen. Store at 4 °C . 1 can be reused.

ECL chemiluminescence reagent
The promage product is divided into two reagents, A and B. When used, it is mixed in equal volume . The volume of the mixture is 1 ml per membrane .

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