Preparation of related reagents for Western Blot experiments

30% (w/v) acrylamide solution

Acrylamide 29g

Methylene bisacrylamide 1g

Add deionized water, dilute to 100 ml, adjust the pH of the solution to less than 7.0, store in a brown bottle at room temperature or 4 °C.

10% SDS

SDS 10g

Distilled water to 100ml

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.

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.

0.5 mol/L Tris·HCl (pH 6.8)

Tris (MW121.14) 15.14g

Ultra pure water 200ml

After dissolving, adjust the pH to 6.8 with concentrated hydrochloric acid, and finally make up to 250 ml with ultrapure water and store at room temperature.

10% ammonium persulfate (AP)

Ammonium persulfate   0.1g

Ultrapure water 1.0ml

After dissolution, it was stored at 4 ° C in the dark and stored for 1 week. (You can first put the persulfate amine dry powder into the EP tube, put a few tubes at a time, add ultrapure water when using)

20% Tween20

Tween20 20ml

Distilled water to 100ml

After mixing, store at 4 °C.

1.74mg/ml (10mmol/L) PMSF

PMSF 0.174g

Isopropyl alcohol 100ml

After dissolving, it was dispensed in a 1.5 ml centrifuge tube and stored at -20 °C.

Single detergent lysate (PMSF)

1mol/L Tris·HCl (pH8.0) 2.5ml

NaCl 0.438g

TritonX-100 0.5ml

Distilled water to 50ml

After mixing, store at 4 °C. At the time of use, PMSF was added to a final concentration of 100 μg/ml (0.87 ml of lysate was added to 1.74 mg/ml of PMSF 50 μl).

RIPA Lysis Formula

50 mM Tris-HCl pH 7.4

150 mM NaCl

1 mM PMSF (plus before use)

1 mM EDTA

1% Triton x-100

1% sodium deoxycholate

0.1% SDS

G250 Coomassie Brilliant Blue Solution (for protein quantification)

Coomassie Brilliant Blue G250 100mg

95% ethanol 50ml

Phosphoric acid 100ml

Distilled water to 1000ml

When preparing, first dissolve the Coomassie blue dye with ethanol, add phosphoric acid and water, mix well, filter with filter paper, and store at 4 °C.

100mg/ml bovine serum albumin (BSA)

BSA 0.1g

0.15 mol/L NaCl 1ml

Store at -20 ° C after dissolution. When preparing the protein standard curve, it was diluted 100-fold with 0.15 mol/L NaCl to 1 mg/ml and stored at -20 °C.

Reduced 5X SDS Loading Buffer  

0.5 mol/L Tris·HCl (pH 6.8) 2.5 ml

Dithiolitol (DTT, MW154.5) 0.39g

SDS 0.5g

Bromophenol blue 0.025g

Glycerin 2.5ml

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

Electrophoresis fluid buffer  

Tris (MW121.14) 3.03g

Glycine (MW75.07) 18.77g

SDS 1g

Distilled water to 1000ml

After dissolution, it can be stored at room temperature, and the secondary solution can be reused 3 to 5 times.

(The electrophoresis fluid can be recycled and reused. Generally, the recovered electrophoresis fluid is added to the lower half of the vertical electrophoresis tank, and the upper half is preferably freshly used.)

(Can be prepared into 10× running buffer for storage and diluted 10 times)

Transfer buffer   

Semi-dry

24mmol/L Tris (MW: 121.14) 1.4536 g

192mmol/L glycine (MW: 75.07) 7.20672 g

20% methanol 100 ml

Distilled water to 500ml

Impregnation (wet rotation)

48mmol/L Tris 2.375g

39mmol/L glycine 11.25g

20% methanol 200ml

0.0375% SDS 0.375g

Distilled water to 1000 ml

After dissolving, it can be stored at room temperature. The secondary solution can be reused 3 to 5 times (the solution is best stored in a 4 degree refrigerator, and the maximum use is about 5 times, otherwise the transfer efficiency is affected). (First dissolve the glycine, Tris and SDS with distilled water, then add methanol, and finally make up the liquid. If you add methanol first, it is difficult to dissolve glycine, Tris and SDS)

(It can be prepared as 2× transfer buffer for storage and used after dilution)

10X Li Chunhong dyeing solution

Li Chunhong S 2g

Trichloroacetic acid 30g

Sulfosalicylic acid 30g

Distilled water to 100ml

TBS buffer

1 mol/L Tris·HCl (pH 7.5) 10ml

NaCl 8.8g

Distilled water to 1000ml

(It can be prepared as 10×TBS buffer for dilution and 10 times diluted)

TBST buffer  

20% Tween20 1.65ml

TBS 700ml

It can be used after mixing, and it is best to use it now.

Blocking solution  

Skimmed milk powder (domestic) 5g

TBST 100ml

It is best to use it now.

Eluting antibody buffer

14.4 mol/L β-mercaptoethanol 700 μl (ventilated 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. Can be reused once. (can be used)

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