secretariatwestern blotting

Western Blotting

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①

Prepare the following reagents in advance

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1×

Running buffer (1L)

:

1×

Transbuffer (1L):

components

Tris

Glycine

SDS

M

（

g

）

3.02 g

14.4 g

1.0 g

components

Tris

Glycine

M

（

g

）

3.02 g

14.4 g

200mL

（

Configure it when in use, -20

℃

refrigerator pre- cooling

）

甲醇

(

Methanol

)

?

1×

TBS

→Weigh 2.42g of Tris, 8g of NaCl, add 800ML of ddH2O

water to stir and dissolve,

adjust the pH to 7.4 with concentrated HCl, then dilute to 1L with three distilled water,

autoclave at high temperature and store at room temperature.

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TBST

(0.1%v/v

Tween 20

in TBS) :TBST (0.1% v/v Tween 20 in TBS): Add 1 mL of

Tween-20 to 1000 mL of 1×

TBS (take it with a cut blue gun head and repeatedly blow it)

(mix it upside down) and store at room temperature.

（

Configure it when in use

）

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10% SDS

：

100 mg SDS is contained in 1 ml of solution, which is prepared with ddH2O.

Store at room temperature.

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10%APS

: 0.1g ammonium persul fate

（

APs

）

< br>+1mLddH2O

（

Configure it when in use

）

?

30% Arc- Bis(29:1)

：

3g Acr- Bis(29:1)+10mLddH2O

（

stored at 4

℃

）

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牛奶封闭液

/

Block buffer(5%w/v drymilk in TBST):5g skim milk powder is dissolved in

100mL TBST, fully mixed, and stored at 4

℃

。

?

抗体稀释液

/

Antibody dilution

：

primary antibody

with 5% BSA (5g dissolved in 100mL

ddH2O), secondary antibody and total primary antibody secondary antibody, GAPDH

primary antibody with 5% Milk, (5% skim milk, with TBST, phosphorylation The protein is

formulated with 5% BSA, ddH2O.)

（

But this one we bought from the reagent dealer

）

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ECL

显影液

:Before use, take ECL developer A and B 1ml respectively, mix and shake to

mix.

②

Preparation process for your sample

Sample collection

Prepare cell lysate in advance to prepare Lysis Buffer containing protease inhibitor. For example,

1 mL of RIPA Lysis Buffer was added to 1.0 ml of cell lysate containing 10 μL of 100 mM PMSF

(final concentration of PMSF was 1 mM).

（

Configure it when in use

）

.

And placed on ice.

a.

In the cell experiment, protein extraction requires a large number of cells. Generally, I will use

a six-well plate for experiments. When the cells grow to 70-80%, each group takes one hole to

extract the protein in the cells. The specific steps are: Pour off the culture solution and pipette the

remaining culture solution

b.

Add 1-2 mL of pre-cooled PBS at 4 °

C per dish. The cells were washed gently by shaking for 1

min, then the washing solution was discarded and washed twice. This step was mainly to wash

away the protein in the medium and reduce the influence on the experimental results.

c.

Add 300 μL of pre

-cooled RIPA Lysis Buffer (containing protease inhibitor) to each well of a

six-well plate, shake the six-well plate repeatedly, lyse the cells well, lyse for about 3 to 5 minutes,

then transfer the cells and lysate to the gun. Pre-cooled in a 1.5 ml centrifuge tube. (

The whole

operation should be carried out on ice as much as possible.

) Continue to lyse the cell lysate on

ice for 30 min. In order to fully lyse the cells, shake or shake frequently.

d.

Centrifuge at 12000 × g for 10 min at 4 ° C, and take 150 μL of the supernatant. (pre

-cooling

the centrifuge in advance)

(here as much as possible to get the supernatant)

e.

After

obtaining

the

protein-rich

supernatant,

the

protein

concentration

of

each

group

was

determined using a BCA protein assay kit. After obtaining each group of protein concentrations,

the

lowest

protein

concentration

group

was

selected,

and

the

remaining

group

protein

concentration was diluted to the lowest concentration of the protein concentration group to reduce

the experimental error. (Because the total amount of protein extracted in the same cell number is

the same, because we adjust the concentration of each group to be consistent, it is convenient to

load, generally the protein loading per well in WB is 20ug~40ug, each in the gel The amount of

protein

added

to

the

well

should

be

consistent).

All

experiments

need

to

be

done

on

ice.

The

complete protein concentration determination process is attached later.

f.

After

adjusting

the

protein

concentration

of

each

group,

add

appropriate

volume

of

5×

SDS

loading

buffer

according

to

each

group

of

protein

solution,

change

5×

SDS

loading

buffer

into

1×

SDS, mix and boil

water for 5 min. The appropriate amount is dispensed in a 200μLEP tube

and

stored

at

-80°

for

readying

to

use.

Avoid

repeated

freeze-thaw

proteins

leading

to

protein

degradation.

③

Preparation of SDS-PAGE gel

（

you

can

prepare

it

in

advance,

save

it

in

electrophoresis

solution,

and

use

it

within

one

week.

）

I usually start WB at noon on a certain day.

(1) Check for adequate,

clean spacers (composite plates), combs (combs), and shelves.

(2) Calculate the concentration of the gel according to the molecular weight of the original antigen

corresponding to the antibody to be detected, and determine the concentration of the gel according

to the molecular weight of the target protein detected.

*Add

1

mL

of

isopropanol

to

the

top

of

the

separation

gel.

After

the

separation

between

the

separation gel and isopropanol, a clear boundary is observed. The separation gel has agglutinated.

This process takes about 45 minutes. Pour out the isopropanol and wash it with ddH2O. In order

to remove the isopropyl alcohol, the upper layer gel, that is, the concentrated gel, is prepared, and

the comb prepared in advance is inserted. After standing for about 45 minutes, the concentrated

gel can be solidified.

1.

Preparation

of

10%

gel

polymerization

catalyst/APS:

For

example,

0.1

g

of

a

gel

polymerization catalyst was weighed, dissolved in water, and made up to 1 ml, which is a 10% gel

polymerization catalyst.

2. Select the appropriate gel concentration according to the molecular weight of the target protein,

and then prepare the SDS-PAGE separation gel (ie the lower layer gel) according to the following

table:

The best separation range of different concentrations of SDS-PAGE separation gel:

SDS-PAGE separation gel concentration

6%gel

8%gel

10%gel

12%gel

15%gel

ingredient

6%gel

ddH2O

30%Acr- Bis(29:1)

1M Tris, pH8.8

10%SDS

10%APS

TEMED

ingredient

8%gel

ddH2O

30%Acr-Bis(29:1)

1M Tris, pH8.8

10%SDS

10%APS

TEMED

Volume

of

each

component

required

to

prepare

different

volumes

of

SDS- PAGE

separation gel (ml)

Volume

of

each

component

required

to

prepare

different

volumes

of

SDS-PAGE

separation gel (ml)

optimal separation range

50-150kD

30-90kD

20-80kD

12-60kD

10-40kD