TITLE: Western Blot
AUTHORS: Beatriz E Nielsen

[DESCRIPTION]
This protocol describes the solutions and steps for performing western blots.

[STEPS]
SECTION: Preparation of lysate from tissues
1. Dissect the tissue with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases.
    
 


SECTION: Preparation of lysate from tissues
2. Place the pieces of tissue microcentrifuge tubes and store samples at -80 °C for later use or keep on ice for immediate homogenization.

SECTION: Preparation of lysate from tissues
3. 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 5 min 
 

 

SECTION: Preparation of lysate from tissues
4. Centrifuge 12000 rpm, 20 min, 4 °C in a microcentrifuge. Remove the tubes from the centrifuge and place on ice, and use the cleared supernatant to determine protein concentration.

SECTION: Protein determination
5. Follow the manufacterer's instructions for protein determination (microplate assay).

  

SECTION: Electrophoresis (SDS-PAGE)
6. Gel preparation:
SECTION: Electrophoresis (SDS-PAGE)
6.1. 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.
 
Protein sizeGel percentage4 – 40 kDa20%12 – 45 kDa15%10 – 70 kDa12.5%15 – 100 kDa10%25 – 100 kDa8%
 
Focusing gel7.5 %10 %12 %15 %# gels24241212Vf10 mL20 mL10 mL20 mL10 mL20 mL10 mL20 mLAcrylamide 30%2.50 mL5.00 mL3.33 mL6.67 mL4.00 mL8.00 mL5.00 mL10.0 mL1.5 M Tris pH=8.92.50 mL5.00 mL2.50 mL5.00 mL2.50 mL5.00 mL2.50 mL5.00 mL10% SDS100 uL200 uL100 uL200 uL100 uL200 uL100 uL200 uLWater4.79 mL9.58 mL3.96 mL7.91 mL3.29 mL6.58 mL2.29 mL4.58 mL 10% APS100 uL200 uL100 uL200 uL100 uL200 uL100 uL200 uLTEMED10 uL20 uL10 uL20 uL10 uL20 uL10 uL20 uL

SECTION: Electrophoresis (SDS-PAGE)
6.2. Stacking gel (upper or top gel):
 
     Stacking gel        # gels      2      4        Vf      5 mL      10 mL        Acrylamide 30%       670 uL      1.33 mL        0.5 M Tris pH=6.8                      10% SDS      50 uL      100 uL        Water      2.99 mL      5.98 mL          10% APS      37.6 uL      75 uL        TEMED      7.5 uL      15 uL   

SECTION: Electrophoresis (SDS-PAGE)
7. Loading and running:
SECTION: Electrophoresis (SDS-PAGE)
7.1. Load equal and appropriate amounts of protein into the wells of the SDS-PAGE gel, along with molecular weight marker.
SECTION: Electrophoresis (SDS-PAGE)
7.2.  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).
SECTION: Transfer
8. 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)    . 
SECTION: Transfer
9. Assemble gel and membrane sandwich: place gel and membrane between buffer-soaked filter papers and sponges. Gently remove air bubbles.
SECTION: Transfer
10. Perform transfer: place transfer assemble in transfer cell and fill with buffer. The time and voltage of transfer require some optimization.
SECTION: Immunodetection
11. Block the membrane for 60 min at Room temperature or   overnight at 4 °C using blocking buffer (5 % volumein TBS-T 0.1% Tween).

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

SECTION: Immunodetection
13. Wash the membrane in three washes of TBS-T, 5 min each.

SECTION: Immunodetection
14. Incubate the membrane with the recommended dilution of horseradish peroxidase HRP-conjugated secondary antibody in blocking buffer 60 min Room temperature

SECTION: Immunodetection
15. Wash the membrane in three washes of TBS-T, 5 min each.
SECTION: Immunodetection
16. For signal development, follow the kit manufacturer’s recommendations:

 
SECTION: Immunodetection
17. Acquire image using darkroom development techniques for chemiluminescence. 
SECTION: Analysis
19. Perform densitometry analysis of the images in Fiji (ImageJ).
SECTION: Buffers
20. 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.
SECTION: Immunodetection
18. If stripping and reprobe is needed:

SECTION: Immunodetection
18.1. Incubate membrane with stripping buffer up to 10 min 

SECTION: Immunodetection
18.2. Wash the membrane in three washes of TBS-T, 5 min each.
SECTION: Immunodetection
18.3. Repeat steps from 11 to 17 with different antibodies or dilutions.