Kỹ thuật Western blot

Western Blot

I. Protein extraction.

Preparation of extraction buffer for V=50mL:

Tris-HCl pH=8	final conc. 60mM	200mM=15mL 500mM=6mL
SDS	2%	10%=10mL 20%=5mL
Sucrose	15%	7.5g
miliQ water up to 50mL		Vtot=50mL

Mix well and let at RT for using to avoid precipitation (fresh use)

Add inhibitor 50X

- Grind sample in liquid N into fine powder

- Add ~ 400ul extraction buffer

- Centrifuge 15 000 rpm for 10min at 4⁰C

- Transfer supernatant into new tube, quantify and store at -80 for later using

II. Quantification:

Prepare mixture of reagent A+B with ratio 50:1 by volume (50 vol.A+1 vol.B) in a falcon covered by aluminum foil.

- Aliquot 200ul mixture (A+B) into wells:

First row for standard: well A to I and blank

Standard	Concentration (ug/mL)
A	2000
B	1500
C	1000
D	750
E	500
F	250
G	125
H	25
I	0
Blank	0

Sample #

- Add 25ul standard conc. A to I, 25ul water for blank well

- Add 23ul miliQ + 2 ul sample (V=25ul) for each sample

- Incubate well array for 20 min at 37⁰C

- Read single wavelength at 562nm in nanodrop

III. Preparation sample for loading and electrophoresis:

10ul of loading sample = V sample + V H₂O + 0.75ul DTT0.8M 20X + 3.75ul loading dye. Protein conc. = 50ug/ul each well so prepare 15ul for each sample volume

Mixture will be pre-heated at 95⁰C (ready at 95⁰C) for denaturation for 5 min

Electrophoresis buffer for 500mL (1L for 2 gels): - 0.5g sodium bisulfite

                                                                                - 100mL 5x running buffer

                                                                                - dH₂O up to 500mL

- Prepare the gel assembly

- Place the gel (20 wells with 10uL well) into electrophoresis machine

- Load 5uL marker proteins into first well

- Load samples # 10uL

- Load 5ul marker proteins close to the last sample well.

- Run at 175mA current or 200V for 1 gel.

IV. Prepare materials for transferring

- Whatman paper: 8 pieces in size of 12.5 x 7 cm

- PVDP membrane

- After finishing electrophoresis: transfer the gel into a tray which containing transferring buffer

- Start saturation the PVDP membrane in:

(1) Methanol and shaking: 2 min

(2) Water: 2 min

(3) Transfer buffer: 10 min

- Wet the surface of the transfer tank with transfer buffer (~100mL) and place the wet membrane on

- Cleaning the gel and cut wells off

- Wet the surface of machine

- Put 4 wet whatman papers (wet by transfer buffer) (12.5x7cm) on surface then

- Membrane with a cut at corner for orientation

- Put the gel on the membrane then

- 4 wet whatman papers

*Note: avoid bubbles at these steps

- Wipe out the extra buffer outside the sandwich with tissue

- Cover the lid and run for 70 min at 97.5mA, V 200, W 100

- Saturate the membrane by ~100mL buffer (1 TTBS 10X buffer: 10 milk v:v)

- Shaking for 30 min

- Washing for 10 min in 1X TBBS buffer

1^st antibody solution: Shaking and incubating for 2h

- Decant the 1^st antibody (re-use ~3 times)

- Wash with washing buffer for 3 min

- Transfer the membrane into a new tray containing washing buffer and shaking for 15 min

- Wash 3 times more with washing buffer 5 min for each time.

2^nd antibody solution: Shaking and incubating for 1h

- Wash with TBBS buffer as done for 1^st antibody solution.

- The membrane can be remained in TBBS buffer unstill the detection process is ready or store in 1X TBS at 4⁰C (the membrane should be immersed completely)

- Detection: ratio 1:1 luminol enhancer: peroxide buffer (V_tot~3ml for each membrane)

- Place the membrane on a tray (flat) and drop detection solution evenly on the surface of the membrane, covering the entire surface

- Cover the membrane by 2 plastic sheets, avoid bubbles

- Visualize in image document system:

- The door open: on chip integration

                        Second 0.2~0.3

                        Preview………capture

                        Close the door…….save

Dynamic……..80 images……..interval  20s

Lights off

Capture …..auto…..save the best one

- Take the image at the best force and condition

Close the door……

- Set up parameters for image docmument