Western Blot
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We have listed the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should produce similar outcomes when using these protocols. However, please be aware that your protocol may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or recommend specific products or equipment. Inclusion of this information is solely for transparency intended to support reproducibility in science.
Abstract
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.
Steps
Lyse cells
Centrifuge µL for 0h 5m 0s at 100x g,0h 0m 0s.
Gently remove supernatant.
Resuspend cell pellet in 1mL of 1X PBS and transfer to a microcentrifuge tube.
Centrifuge for 0h 5m 0s at 100x g,0h 0m 0s.
Carefully remove supernatant.
Resuspend cell pellet in appropriate volume of cold lysis buffer.
Incubate on ice for 0h 30m 0s.
Centrifuge lysate for 0h 15m 0s at 14.000x g,0h 0m 0s at 4°C.
Transfer supernatant to a clean microcentrifuge tube.
Use the lysate immediately or store at -80°C until ready to use.
Determine the total protein concentration and prepare the lysate for SDS-PAGE
Determine the protein concentration using a Pierce BCA Assay Kit or other preferred method for protein determination.
Prepare a 50:1 Reagent A to Reagent B dilution of the BCA assay.
Prepare serial dilutions of the BSA standard that range from 0–2000 µg/mL.
In duplicate, dilute 10µL of standard, blank, and lysate samples into 200µL of BCA reagent in a 96-well microtiter plate.
Incubate for 0h 30m 0s at 37°C.
Determine the absorbance at 590 nm.
Calculate the average absorbance of the duplicate samples on the plate.
Subtract the average absorbance of the blank from all of the samples.
Plot a standard curve of BSA standard concentration versus absorbance.
Extrapolate the total protein concentration of the sample from the standard curve.
Determine the volume of sample required to load an equivalent amount of total protein for each sample.
Prepare sample for loading as follows:
Add 10% v/v β-mercaptoethanol to the 4X protein loading buffer.
Dilute 4X protein loading buffer in the sample to 1X.
Boil the samples for 0h 10m 0s at 100°C.
SDS-PAGE
Prepare the precast gel as follows:
Remove the gel from the plastic packaging.
Remove tape and plastic comb.
Rinse the wells with deionized water 3x.
Shake the gel gently between washes to remove residual water.
Load the gel into the chamber of the SDS-PAGE gel tank.
Close the clamp.
Prepare 1X running buffer as follows:
Dilute 25mL of 20X running buffer to 500mL with deionized water. Mix well.
Fill the chamber of the SDS-PAGE gel tank with the 1X running buffer.
Load the samples into the gel.
Load 5–10 µL of the prestained protein ladder.
Place the lid on the tank and plug the electrode cords into the power supply.
Run the gel at 100 V for 10–15 min or until the samples have moved out of the wells and into the gel.
Increase the voltage to 150 V and continue running the gel until you have obtained the desired separation.
Gently open the gel with the spatula.
Dry transfer
Soak the gel for 0h 15m 0s in 20% ethanol in deionized water.
To prepare 20% ethanol, dilute 2mL of ethanol into 8mL of deionized water and mix well.
Assemble the transfer sandwich as follows:
Unseal the iBlot 2 PVDF Mini transfer stack.
Remove air bubbles using the roller.
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.
Close the lid of the device.
Select the desired method and make sure the parameters are correct.
Transfer for 5–6 min for proteins <30 kDa.
Transfer for 8–10 min for proteins >150 kDa.
Select Start Run.
Set the Top Stack to one side and discard the white separator.
Keep the Bottom Stack in the plastic tray.
Place the Bottom Stack on the blotting surface.
Align the electrical contacts on the blotting surface of the iBlot 2 Gel Transfer Device.
Wet the pre-run gel in deionized water and place it on the transfer membrane of the bottom stack.
Soak a piece of iBlot Filter Paper in deionized water.
Place the pre-soaked iBlot Filter Paper on the gel and remove the air bubbles with the roller.
Place the Top Stack over the pre-soaked filter paper.
When the run is complete, select Done.
Blocking
Prepare 1X TBST as follows:
25mL of 20X TBS
2.5mL of Tween-20
472.5mL of deionized water
Mix well
Prepare blocking buffer as follows:
Dilute 5% w/v non-fat milk into 1X TBST. (Example: 5g of non-fat milk into 100mL of 1X TBST.)
Remove the membrane from the transfer device and place it in a container with 1X TBST, protein side up.
Block the membrane in blocking buffer for 1h 0m 0s at Room temperature on a shaking platform.
Wash the membrane 3x for 0h 5m 0s in 1X TBST at Room temperature on a shaking platform.
Antibody incubation
Dilute the primary antibody to the desired concentration in blocking buffer.
Incubate the membrane overnight in primary antibody at 4°C on a rocking platform.
Wash the membrane 3x for 0h 5m 0s in 1X TBST at Room temperature on a shaking platform.
Dilute the horseradish peroxidase-conjugated secondary antibody to the desired concentration in blocking buffer.
Incubate the membrane with secondary antibody for 1h 0m 0s at Room temperature on a shaking platform.
Wash the membrane 3x for 0h 5m 0s in 1X TBST at Room temperature on a shaking platform.
Prepare the chemiluminescence substrate by mixing 1:1 reagent A to reagent B.
Gently incubate the membrane in the chemiluminescence reagent for 0h 5m 0s at Room temperature.
Cover the membrane with clear plastic and use a gel imager with chemiluminescence detection or a dark room to detect the bands.
