TITLE: Western Blot
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
Western blot is a technique used to separate proteins by size followed by detection using antibodies specific to the protein of interest. This protocol describes the basic steps for lysing cells, determining total protein concentration in the lysate, running a precast SDS-PAGE gel, and immunoblotting.

[BEFORE_START]
Refer to the manufacturer's instructions for additional information specific to your antibody, such as ideal blocking buffer and optimal antibody concentrations. Consider titrating your antibody to determine the optimal dose.

Secondary antibodies must match the host species of the primary antibody. For example, use an anti-mouse secondary antibody for primary antibodies raised in a mouse.

[GUIDELINES]
General Considerations
Acrylamide percentage varies in SDS-PAGE gels. Use lower  percentage acrylamide when immunoblotting high molecular weight proteins and higher percentage acrylamide when immunoblotting low molecular  weight proteins.

This protocol uses a dry transfer device but can be adapted for wet and semi-dry transfer approaches.

Workflow Timeline
Day 1: Prepare lysates, run SDS-PAGE, transfer, block, incubate with primary antibody
Day 2: Incubate with secondary antibody

Tips and Troubleshooting:
The optimal lysis buffer will vary depending on the sample type and cellular location of the protein of interest. You may  need to try a variety of lysis buffers to find the best for your target.

The optimal concentration and blocking buffer will vary between antibodies. Review the instructions before starting your experiment and consider titrating your antibody to determine the optimal dose.

To ensure that your antibody is both functioning as expected and is specific, include a positive control sample that you know expresses the protein, such as cells transfected with the protein of interest, and a negative control sample that does not express the protein of  interest.

[STEPS]
SECTION: Lyse cells
1. Centrifuge   for 5 min at 100 x g.


SECTION: Lyse cells
2. Gently remove supernatant.
SECTION: Lyse cells
3. Resuspend cell pellet in 1 mL of 1X PBS and transfer to a microcentrifuge tube.

SECTION: Lyse cells
4. Centrifuge for 5 min at 100 x g.


SECTION: Lyse cells
5. Carefully remove supernatant.
SECTION: Lyse cells
6. Resuspend cell pellet in appropriate volume of cold lysis buffer.
 

 

SECTION: Lyse cells
7. Incubate on ice for 30 min.

SECTION: Lyse cells
8. Centrifuge lysate for 15 min at 14.000 x g at 4 °C.

SECTION: Lyse cells
9. Transfer supernatant to a clean microcentrifuge tube.
SECTION: Lyse cells
10. Use the lysate immediately or store at -80 °C until ready to use.

SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
11. Determine the protein concentration using a Pierce BCA Assay Kit or other preferred method for protein determination.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
12. Determine the volume of sample required to load an equivalent amount of total protein for each sample.
 

SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
13. Prepare sample for loading as follows:
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
14. Boil the samples for 10 min at 100 °C.

SECTION: SDS-PAGE
15. Prepare the precast gel as follows:
SECTION: SDS-PAGE
16. Prepare 1X running buffer as follows:
SECTION: SDS-PAGE
17. Fill the chamber of the SDS-PAGE gel tank with the 1X running buffer.
SECTION: SDS-PAGE
18. Load the samples into the gel.
SECTION: SDS-PAGE
19. Load 5–10 µL of the prestained protein ladder.
SECTION: SDS-PAGE
20. Place the lid on the tank and plug the electrode cords into the power supply.
SECTION: SDS-PAGE
21. Run the gel at 100 V for 10–15 min or until the samples have moved out of the wells and into the gel.
SECTION: SDS-PAGE
22. Increase the voltage to 150 V and continue running the gel until you have obtained the desired separation.
SECTION: SDS-PAGE
23. Gently open the gel with the spatula.
SECTION: Dry transfer
24. Soak the gel for 15 min in 20% ethanol in deionized water.

SECTION: Dry transfer
25. Assemble the transfer sandwich as follows:
SECTION: Dry transfer
26. When the run is complete, select Done.
SECTION: Blocking
27. Prepare 1X TBST as follows:
SECTION: Blocking
28. Prepare blocking buffer as follows:
SECTION: Blocking
29. Remove the membrane from the transfer device and place it in a container with 1X TBST, protein side up.
SECTION: Blocking
30. Block the membrane in blocking buffer for 60 min at Room temperature on a shaking platform.

SECTION: Blocking
31. Wash the membrane 3x for 5 min in 1X TBST at Room temperature on a shaking platform.

SECTION: Antibody incubation
32. Dilute the primary antibody to the desired concentration in blocking buffer.
 

SECTION: Antibody incubation
33. Incubate the membrane overnight in primary antibody at 4 °C on a rocking platform.
 


SECTION: Antibody incubation
34. Wash the membrane 3x for 5 min in 1X TBST at Room temperature on a shaking platform.

SECTION: Antibody incubation
35. Dilute the horseradish peroxidase-conjugated secondary antibody to the desired concentration in blocking buffer.
 

SECTION: Antibody incubation
36. Incubate the membrane with secondary antibody for 60 min at Room temperature on a shaking platform.

SECTION: Antibody incubation
37. Wash the membrane 3x for 5 min in 1X TBST at Room temperature on a shaking platform.

SECTION: Antibody incubation
38. Prepare the chemiluminescence substrate by mixing 1:1 reagent A to reagent B.
SECTION: Antibody incubation
39. Gently incubate the membrane in the chemiluminescence reagent for 5 min at Room temperature.

SECTION: Antibody incubation
40. Cover the membrane with clear plastic and use a gel imager with chemiluminescence detection or a dark room to detect the bands.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
11.1. Prepare a 50:1 Reagent A to Reagent B dilution of the BCA assay.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
11.2. Prepare serial dilutions of the BSA standard that range from 0–2000 µg/mL.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
11.3. In duplicate, dilute 10 µL of standard, blank, and lysate samples into 200 µL of BCA reagent in a 96-well microtiter plate.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
11.4. Incubate for 30 min at 37 °C.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
11.5. Determine the absorbance at 590 nm.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
11.6. Calculate the average absorbance of the duplicate samples on the plate.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
11.7. Subtract the average absorbance of the blank from all of the samples.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
11.8. Plot a standard curve of BSA standard concentration versus absorbance.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
11.9. Extrapolate the total protein concentration of the sample from the standard curve.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
13.1. Add 10% v/v β-mercaptoethanol to the 4X protein loading buffer.
SECTION: Determine the total protein concentration and prepare the lysate for SDS-PAGE
13.2. Dilute 4X protein loading buffer in the sample to 1X.
SECTION: SDS-PAGE
15.1. Remove the gel from the plastic packaging.
SECTION: SDS-PAGE
15.2. Remove tape and plastic comb.
SECTION: SDS-PAGE
15.3. Rinse the wells with deionized water 3x.
SECTION: SDS-PAGE
15.4. Shake the gel gently between washes to remove residual water.
SECTION: SDS-PAGE
15.5. Load the gel into the chamber of the SDS-PAGE gel tank.
 

SECTION: SDS-PAGE
15.6. Close the clamp.
SECTION: SDS-PAGE
16.1. Dilute 25 mL of 20X running buffer to 500 mL with deionized water. Mix well.

SECTION: Dry transfer
24.1. To prepare 20% ethanol, dilute 2 mL of ethanol into 8 mL of deionized water and mix well.

SECTION: Dry transfer
25.1. Unseal the iBlot 2 PVDF Mini transfer stack.
SECTION: Dry transfer
25.2. Set the Top Stack to one side and discard the white separator.
SECTION: Dry transfer
25.3. Keep the Bottom Stack in the plastic tray.
SECTION: Dry transfer
25.4. Place the Bottom Stack on the blotting surface.
SECTION: Dry transfer
25.5. Align the electrical contacts on the blotting surface of the iBlot 2 Gel Transfer Device.
SECTION: Dry transfer
25.6. Wet the pre-run gel in deionized water and place it on the transfer membrane of the bottom stack.
SECTION: Dry transfer
25.7. Soak a piece of iBlot Filter Paper in deionized water.
SECTION: Dry transfer
25.8. Place the pre-soaked iBlot Filter Paper on the gel and remove the air bubbles with the roller.
SECTION: Dry transfer
25.9. Place the Top Stack over the pre-soaked filter paper.
SECTION: Dry transfer
25.10. Remove air bubbles using the roller.
SECTION: Dry transfer
25.11. Place the Absorbent Pad on top of the Top Stack such that the electrical contacts are aligned with the corresponding electrical contacts on the blotting surface of the iBlot 2 Gel Transfer Device.
SECTION: Dry transfer
25.12. Close the lid of the device.
SECTION: Dry transfer
25.13. Select the desired method and make sure the parameters are correct.
SECTION: Dry transfer
25.14. Transfer for 5–6 min for proteins <30 kDa.
SECTION: Dry transfer
25.15. Transfer for 8–10 min for proteins >150 kDa.
SECTION: Dry transfer
25.16. Select Start Run.
SECTION: Blocking
27.1. 25 mL of 20X TBS

SECTION: Blocking
27.2. 2.5 mL of Tween-20

SECTION: Blocking
27.3. 472.5 mL of deionized water

SECTION: Blocking
27.4. Mix well
SECTION: Blocking
28.1. Dilute 5% w/v non-fat milk into 1X TBST. (Example: 5 g of non-fat milk into 100 mL of 1X TBST.)