Western Blot

Dissect the tissue with clean tools, On ice preferably, and as quickly as possible to prevent degradation by proteases.

Place the pieces of tissue microcentrifuge tubes and store samples at -80°C for later use or keep on ice for immediate homogenization.

Add ice-cold lysis buffer rapidly to the tubes and homogenize with an electric homogenizer. Then denature the samples at 100°C in a hot plate for 0h 5m 0s

Centrifuge 12000rpm,4°C in a microcentrifuge. Remove the tubes from the centrifuge and place on ice, and use the cleared supernatant to determine protein concentration.

Follow the manufacterer's instructions for protein determination (microplate assay).

Gel preparation:

Focusing gel (lower or bottom gel):

Prepare the focusing or resolving gel with the percentage or concentration required, depending on the size of your protein of interest. Gradient gels can also be used.

A	B
Protein size	Gel percentage
4 – 40 kDa	20%
12 – 45 kDa	15%
10 – 70 kDa	12.5%
15 – 100 kDa	10%
25 – 100 kDa	8%

A	B	C	D	E
Focusing gel	7.5 %	10 %	12 %	15 %
Vf	10 mL	10 mL	10 mL	10 mL
Acrylamide 30%	2.50 mL	3.33 mL	4.00 mL	5.00 mL
1.5 M Tris pH=8.9	2.50 mL	2.50 mL	2.50 mL	2.50 mL
10% SDS	100 uL	100 uL	100 uL	100 uL
Water	4.79 mL	3.96 mL	3.29 mL	2.29 mL
10% APS	100 uL	100 uL	100 uL	100 uL
TEMED	10 uL	10 uL	10 uL	10 uL

Stacking gel (upper or top gel):

A	B
Stacking gel
Vf	5 mL
Acrylamide 30%	670 µL
0.5 M Tris pH=6.8	1.25 mL
10% SDS	50 µL
Water	2.99 mL
10% APS	37.6 µL
TEMED	7.5 µL

Loading and running:

Load equal and appropriate amounts of protein into the wells of the SDS-PAGE gel, along with molecular weight marker.

Run the gel for 1–2 h at 100 V (the time and voltage may require optimization, check the marker to obtain an optimal separation of the bands with the target molecular weight).

Equilibrate gel and membrane in transfer buffer (membrane can be nitrocellulose or PVDF, previosuly activated with methanol for 1 min and rinsed in transfer buffer) .

Assemble gel and membrane sandwich: place gel and membrane between buffer-soaked filter papers and sponges. Gently remove air bubbles.

Perform transfer: place transfer assemble in transfer cell and fill with buffer. The time and voltage of transfer require some optimization.

Block the membrane for 1h 0m 0s at Room temperature or overnight at 4°C using blocking buffer (5% volumein TBS-T 0.1% Tween).

Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer at 4°C overnight.

Wash the membrane in three washes of TBS-T, 0h 5m 0s each.

Incubate the membrane with the recommended dilution of horseradish peroxidase HRP-conjugated secondary antibody in blocking buffer 1h 0m 0s Room temperature

Wash the membrane in three washes of TBS-T, 0h 5m 0s each.

For signal development, follow the kit manufacturer’s recommendations:

Acquire image using darkroom development techniques for chemiluminescence.

If stripping and reprobe is needed:

Incubate membrane with stripping buffer up to 0h 10m 0s

Wash the membrane in three washes of TBS-T, 0h 5m 0s each.

Repeat steps from 11 to 17 with different antibodies or dilutions.

Perform densitometry analysis of the images in Fiji (ImageJ).

10X STE buffer

• 100 mM Tris-Cl: pH=7.5

• 10 mM EDTA pH=8.0

• 10% SDS. 10X TBS (Tris-buffered saline)

• 200 mM Tris.

• 1500 mM NaCl.

• pH=7.6 with 12 N HCl. 1X TBS

• 20 mM Tris.

• 150 mM NaCl.

• pH=7.6 with 12 N HCl.

TBS-T (Tris-buffered saline, 0.1% Tween-20)

• 20 mM Tris.
• 150 mM NaCl.
• 0.1% Tween-20.
• pH=7.6 with 12 N HCl.