Preparation of basic reagents for Western Blot

Protein extraction reagent

RIPA lysate:

50 mM Tris (MW121.14, pH 7.5) 3.02 g

150 mM NaCl 4.38 g

1% TritonX-100 5ml

1% sodium deoxycholate 5 g

0.1% SDS 0.5 g

Distilled water to 500ml.

Adjust the pH to 7.5.

Mix 4 ? after storage, long-term preservation at -20 ?. When used, add the protease inhibitor cocktail.

 

Protease inhibitor cocktail

Required mother solution ingredients:

(1) 100 mmol / L PMSF

PMSF 17.4 mg

Isopropyl alcohol 1ml

After dissolution, the mixture was dispensed into a 1.5 ml centrifuge tube and stored at -20 ° C.

(2) 10 mM leupeptin leupeptin: stored at -20 ° C

(3) 2.1 mg / ml Aprotinin: 4 ° C

Preparation: the final concentration of ingredients per ml of lysis solution by the amount of mother solution

PMSF 1 mmol / L (storage concentration diluted 100 times) 10ul

Leupeptin 0.1 mmol / L (100 ?l diluted)

Aprotinin 1ug / ml (storage concentration diluted 2000 times) 0.5ul

Each component was add directly to the RIPA lysate used, followed by extraction of the protein at 60 ul / well (12-well plate).

 

Protein Quantitative Reagents

G250 Coomassie brilliant blue solution (protein quantitative)

Coomassie brilliant blue G250 100mg

95% ethanol 50ml

85% phosphoric acid 100ml

Distilled water to 1000ml

Preparation, the first dissolved in ethanol Kaomasi bright blue dye, then add phosphoric acid and water, mix, filter with filter paper, 4 ? stored in a brown bottle.

Bovine serum albumin BSA stock solution (1 mg / ml):

100mg BSA powder dissolve in 20 ml double distilled water, constant volume to 100ml, add a small amount of preservatives, 4 ? preservation.

 

Loading reagent

1.4X loading buffer

1.0 mol / L Tris · HCl (pH 6.8) 2 ml

SDS 0.8 g

Bromophenol blue 0.04 g

Glycerol 4ml

Deionized water to 10ml.

After mixing, the mixture was dispensed into a 1.5 ml centrifuge tube and stored at 4 ° C. Diluted to 1X when used.

 

1.0 mol / L dithiothreitol (DTT) (10X)

Dissolve 3.09 g DTT with 20 ml 0.01 mol / L sodium acetate solution (pH 5.2) and dispensing into 1 ml of small portions at -20 ° C. Diluted to 1X when used.

For example: loading amount 20?l – 4X loading buffer 5?l + DTT2?l + sample protein 13?l

 

Working fluid

Gel Making (1-6):

1.0 mol / L Tris · HCl (pH 6.8)

Tris (MW121.14) 12.114g

Distilled water 100ml

After dissolving, adjust to pH 6.8 with concentrated hydrochloric acid and store at room temperature.

5 mol / L Tris · HCl (pH 8.8)

Tris (MW121.14) 18.671g

Distilled water 100ml

After dissolving, adjust to pH 8.8 with concentrated hydrochloric acid and store at room temperature.

10% SDS

SDS 10 g

Distilled water to 100ml

If difficult in dissolving, it can be dissolved in 50 ? water bath, room temperature preservation.

If there is precipitation after the long-term preservation, please water bath before usage.

 

10% persulfate (AP)

0.1 g of sulfuric acid amine

Distilled water 1ml

(Weigh ammonium persulfate powder and then place in 1.5mlEP tube. Add distilled water when use. 4 ? preservation, preservation time of 1 week)

 

Tetramethylethylenediamine stock solution (TEMED): 4 ° C.

 

30% acrylamide: stored at 4 ° C.

10% Lower glue (two pieces of glue, 15ml):

Sequentially add

Deionized water 5.9ml

30% acrylamide 5ml

1.5M Tris-HCl (pH 8.8) 3.8 ml

10% SDS 150 ?l

10% AP 150 ?l

TEMED 6 ?l

To speed up the gel, you can increase the amount of ammonium persulfate and TEMED, according to the appropriate temperature adjustment at the time of the experiment, adding TEMED after mixing immediately. Add with n-butanol (1ml) to defoam the bubbles.

5% Upper glue (two pieces of plastic, 6ml):

Sequentially add

Deionized water 4.1ml

30% acrylamide 1 ml

1MTris-HCl (pH 6.8) 0.75 ml

10% SDS 60 l

10% AP 60 ?l

TEMED 6 ?l

To speed up the gel, you can increase the amount of ammonium persulfate and TEMED, according to the appropriate temperature adjustment at the time of the experiment, adding TEMED after mixing immediately.

 

7.5X electrophoretic buffer

Tris (MW121.14) 15.1 g

Glycine (MW 75.07) 94 g

SDS 5.00g

Distilled water to 1000ml

Dissolve at room temperature after storage, dilute with 5 times, usually 160ml can be prepared into 800ml.

 

10X transfer membrane buffer

Glycine (MW 75.07) 151.1 g

Tris (MW121.14) 30.3 g

Distilled water to 1000ml

Dissolve at room temperature after storage, diluted with 10 times, and add methanol to 20%.

Usually take 80ml mother liquor, add 560ml distilled water, the most 160ml of methanol, can be prepared into 800ml. (First increase in methanol precipitation)

 

10XTBS buffer

Tris (MW121.14) 24.2 g

NaCl 80.0 g

Distilled water to 1000ml

(Concentrated hydrochloric acid adjusted to pH 7.6), dissolved at room temperature after storage.

1XTBST buffer

10XTBS buffer 100ml

Distilled water 900ml

Tween-20 1 ml

Tween-20 is more viscous, should be slowly drew and can cut off a piece of pipette tip, you can prevent the generation of bubbles.

Dissolved at room temperature after storage.

 

Blocking liquid / antibody dilution (5% skimmed milk):

1XTBST buffer 95-100 ml

Skimmed milk powder 5g

Dissolved 4 ? after storage, can be used within a week.