Western Blotting: A Comprehensive Guide
Western Blotting Procedure and Analysis
Purpose of Western Blotting
Separates proteins on a gel according to molecular weight.
Proteins are then transferred onto a membrane for antibody detection.
Sample Preparation
Two samples from PC12 cells:
Control sample (untreated).
Stimulated sample (treated with nerve growth factor or NGF).
Add blue sample buffer to protein lysates.
Contains reducing agents (e.g., beta-mercaptoethanol) to linearize proteins.
Provides proteins with a negative charge proportional to their size.
Heat samples at 100 degrees Celsius for 2 minutes.
Speeds up denaturation by increasing molecular motion.
More important for membrane samples.
Mini Protein Gel Setup
Remove the gel from packaging.
Remove green tape from the bottom of the cassette.
Remove the comb by pulling upward.
Set electrode assembly to the open position.
Place gel cassette and buffer dam into the electrode assembly with short plates facing inward.
Push gels towards each other and lock them in place with green arms.
Place electrophoresis module into the tank.
Fill buffer chambers with 1x SDS PAGE running buffer, allowing it to overflow.
Sample Loading
Load molecular weight markers and samples into the gel.
Load molecular weight marker into the first lane.
Load control and stimulated samples into adjacent wells, ensuring equal protein amounts.
Connect power pack to the lid of the gel apparatus and place the lid onto the gel tank.
Set power pack to a constant 200 volts for 30 minutes.
Protein Electrophoresis (SDS PAGE)
Charged protein molecules are transported through a solvent by an electric field.
SDS PAGE separates proteins primarily by mass.
SDS denatures and binds proteins, making them uniformly negatively charged.
All SDS-bound proteins migrate towards the positively charged electrode when a current is applied.
Proteins with less mass travel more quickly through the gel due to the sieving effect of the gel matrix.
After electrophoresis, proteins can be detected with stains or transferred to a membrane for Western blotting.
Post-Electrophoresis Steps
Turn off the power supply and disconnect electrical leads.
Remove the lid and gels from the cell.
Discard the running buffer.
Remove the gel from the gel electrode assembly.
Open the cassette using the opening lever; apply downward pressure to break each seal but do not twist the lever.
Pull the two plates apart and gently remove the gel.
Use the other half of the cassette to peel away the lanes from the gel.
Gel Equilibration
Rinse and soak the gel in transfer buffer for approximately 10 minutes.
Removes contaminating electrophoresis buffer salts, which can increase conductivity and heat during transfer.
Soak nitrocellulose membrane and blotting paper in transfer buffer for 10 minutes.
Transfer to Nitrocellulose Membrane
Proteins are transferred from the gel onto a solid support (nitrocellulose membrane).
Suitable for immunological or biochemical analyses (antibody staining and protein detection).
Transfer is performed by passing a current across the gel to the membrane.
The gel and membrane are assembled into a sandwich with filter paper.
Filter paper protects the gel and membrane and ensures close contact.
The membrane is placed between the gel and the positive electrode.
Negatively charged proteins migrate from the gel onto the membrane.
Transfer buffer is similar to SDS PAGE running buffer with methanol, which helps proteins bind to the nitrocellulose membrane.
Use a roller to remove air bubbles between the gel and the membrane.
Semi-Dry Transfer Using Turbo Blotter
The gel, blotting paper, and filter sandwich are assembled onto a larger electrode plate.
An electric field is generated, and the buffer is confined to the stacks of wet filter paper.
Transfer for 7 minutes at a fixed voltage of 25 volts with variable amperage.
After blotting, turn off the power and disassemble the apparatus.
Evaluate transfer success by noting the appearance of pre-stained markers on the blot compared to the gel.
Blocking the Membrane
Block the remaining binding surface of the membrane to prevent nonspecific antibody binding.
Use any protein that does not have a binding affinity for the target.
Commonly use bovine serum albumin (BSA) diluted in TBST or PBST buffers.
Membrane may be cut in half before blocking.
Antibody Incubation
Use two different primary antibodies to detect two different proteins.
Antibodies recognize antigens (proteins) based on their structure and content, binding to the epitope.
Each antibody binds to one unique epitope due to its unique antigen-binding site.
In this experiment, use primary antibodies recognizing total MAP kinase and the activated (phosphorylated) form of MAP kinase.
Incubate the primary antibody for one hour at room temperature.
Washing Steps
Carry out three washes with TBS-Tween (TBST).
Removes excess primary antibody, which may interfere or provide background noise.
Secondary Antibody Incubation
Secondary antibodies bind to the heavy chains of primary antibodies without interfering with antigen binding.
Anti-rabbit IgG conjugated with alkaline phosphatase (produced in goat) is used.
The secondary antibody is made in a different species than the primary antibody (rabbit) and the samples (rat cells).
Minimizes nonspecific binding that leads to false positives and high background noise.
Following incubation, wash blots three times with TBST to remove unbound secondary antibody.
Protein Visualization
Use a substrate called BCIP/NBT.
Alkaline phosphatase (conjugated to the secondary antibody) facilitates the BCIP/NBT system.
Based on the hydrolysis of BCIP and reduction of NBT, producing a deep purple reaction product if the protein of interest is present.
Results and Analysis
Two final blots are probed with total MAP kinase (left) and active MAP kinase antibody (right).
Total MAP kinase blot shows the protein marker, control PC12 cell extract, and stimulated PC12 cell extract.
Both control and stimulated samples contain total MAP kinase at equivalent levels.
Total MAP kinase detection includes both activated and non-activated forms.
Active MAP kinase blot shows the protein marker, control PC12 cell extract, and stimulated PC12 cell extract.
The control sample does not contain activated MAP kinase, while the stimulated sample does.
Conclusion: Treating PC12 cells with NGF results in the activation/phosphorylation of MAP kinase.
Two distinct bands for each sample represent different isoforms of MAP kinase.
Top band: 44 kDa isoform.
Lower band: 42 kDa isoform.
Protein isoforms are proteins similar to each other with similar roles within cells.
A single gene can encode multiple isoforms via alternative splicing (e.g., MAP kinase).
Purpose of Western Blotting
Separates proteins on a gel according to molecular weight. This separation allows for the identification and quantification of specific proteins within a complex mixture.
Proteins are then transferred onto a membrane for antibody detection. The membrane-bound proteins are probed with specific antibodies to detect the protein of interest.
Sample Preparation
Two samples from PC12 cells:
Control sample (untreated).
Stimulated sample (treated with nerve growth factor or NGF). NGF stimulation mimics physiological conditions, allowing for observation of protein activation.
Add blue sample buffer to protein lysates.
Contains reducing agents (e.g., beta-mercaptoethanol) to linearize proteins by breaking disulfide bonds. This ensures consistent protein migration during electrophoresis.
Provides proteins with a negative charge proportional to their size, which is essential for SDS-PAGE.
Heat samples at 100 degrees Celsius for 2 minutes.
Speeds up denaturation by increasing molecular motion, ensuring proteins are fully unfolded.
More important for membrane samples, where protein aggregates may be more resistant to denaturation.
Mini Protein Gel Setup
Remove the gel from packaging.
Remove green tape from the bottom of the cassette to allow ion flow during electrophoresis.
Remove the comb by pulling upward, creating wells for sample loading.
Set electrode assembly to the open position.
Place gel cassette and buffer dam into the electrode assembly with short plates facing inward.
Push gels towards each other and lock them in place with green arms to ensure proper electrical contact.
Place electrophoresis module into the tank.
Fill buffer chambers with 1x SDS PAGE running buffer, allowing it to overflow to ensure continuous ion flow.
Sample Loading
Load molecular weight markers and samples into the gel. Molecular weight markers help in determining the size of the separated proteins.
Load molecular weight marker into the first lane.
Load control and stimulated samples into adjacent wells, ensuring equal protein amounts. Equal protein loading controls for variations in protein expression levels.
Connect power pack to the lid of the gel apparatus and place the lid onto the gel tank.
Set power pack to a constant 200 volts for 30 minutes. The voltage drives the protein migration through the gel.
Protein Electrophoresis (SDS PAGE)
Charged protein molecules are transported through a solvent by an electric field.
SDS PAGE separates proteins primarily by mass.
SDS denatures and binds proteins, making them uniformly negatively charged. This ensures separation is based on size rather than charge.
All SDS-bound proteins migrate towards the positively charged electrode when a current is applied.
Proteins with less mass travel more quickly through the gel due to the sieving effect of the gel matrix. The gel matrix acts as a molecular sieve, impeding larger proteins more than smaller ones.
After electrophoresis, proteins can be detected with stains or transferred to a membrane for Western blotting. Transfer to a membrane allows for antibody-based detection.
Post-Electrophoresis Steps
Turn off the power supply and disconnect electrical leads.
Remove the lid and gels from the cell.
Discard the running buffer.
Remove the gel from the gel electrode assembly.
Open the cassette using the opening lever; apply downward pressure to break each seal but do not twist the lever.
Pull the two plates apart and gently remove the gel.
Use the other half of the cassette to peel away the lanes from the gel to ensure the lanes are properly separated.
Gel Equilibration
Rinse and soak the gel in transfer buffer for approximately 10 minutes. This ensures that the transfer buffer can properly penetrate the gel.
Removes contaminating electrophoresis buffer salts, which can increase conductivity and heat during transfer, potentially distorting the protein bands.
Soak nitrocellulose membrane and blotting paper in transfer buffer for 10 minutes. This prepares the membrane and blotting paper for efficient protein transfer.
Transfer to Nitrocellulose Membrane
Proteins are transferred from the gel onto a solid support (nitrocellulose membrane). The membrane provides a stable surface for subsequent antibody probing.
Suitable for immunological or biochemical analyses (antibody staining and protein detection). Nitrocellulose is chosen for its high protein-binding affinity.
Transfer is performed by passing a current across the gel to the membrane.
The gel and membrane are assembled into a sandwich with filter paper.
Filter paper protects the gel and membrane and ensures close contact, promoting uniform protein transfer.
The membrane is placed between the gel and the positive electrode.
Negatively charged proteins migrate from the gel onto the membrane.
Transfer buffer is similar to SDS PAGE running buffer with methanol, which helps proteins bind to the nitrocellulose membrane. Methanol also helps to remove SDS from the proteins, facilitating binding to the membrane.
Use a roller to remove air bubbles between the gel and the membrane, ensuring even protein transfer.
Semi-Dry Transfer Using Turbo Blotter
The gel, blotting paper, and filter sandwich are assembled onto a larger electrode plate.
An electric field is generated, and the buffer is confined to the stacks of wet filter paper. This setup allows for efficient and rapid protein transfer.
Transfer for 7 minutes at a fixed voltage of 25 volts with variable amperage.
After blotting, turn off the power and disassemble the apparatus.
Evaluate transfer success by noting the appearance of pre-stained markers on the blot compared to the gel. This confirms that proteins have been efficiently transferred from the gel to the membrane.
Blocking the Membrane
Block the remaining binding surface of the membrane to prevent nonspecific antibody binding. Blocking reduces background noise in subsequent steps.
Use any protein that does not have a binding affinity for the target. Common blocking agents include BSA or non-fat dry milk.
Commonly use bovine serum albumin (BSA) diluted in TBST or PBST buffers. BSA is an inexpensive and effective blocking agent.
Membrane may be cut in half before blocking to probe for different proteins separately.
Antibody Incubation
Use two different primary antibodies to detect two different proteins. This allows for simultaneous detection of multiple proteins on the same blot.
Antibodies recognize antigens (proteins) based on their structure and content, binding to the epitope. Epitope-specific binding ensures high target specificity.
Each antibody binds to one unique epitope due to its unique antigen-binding site. This ensures minimal cross-reactivity.
In this experiment, use primary antibodies recognizing total MAP kinase and the activated (phosphorylated) form of MAP kinase. This allows for assessing both total protein levels and activation status.
Incubate the primary antibody for one hour at room temperature to allow sufficient binding to the target protein.
Washing Steps
Carry out three washes with TBS-Tween (TBST) to remove unbound antibodies.
Removes excess primary antibody, which may interfere or provide background noise.
Secondary Antibody Incubation
Secondary antibodies bind to the heavy chains of primary antibodies without interfering with antigen binding. This amplifies the signal and enables detection.
Anti-rabbit IgG conjugated with alkaline phosphatase (produced in goat) is used. The secondary antibody is specific to the species in which the primary antibody was raised.
The secondary antibody is made in a different species than the primary antibody (rabbit) and the samples (rat cells). This reduces non-specific binding.
Minimizes nonspecific binding that leads to false positives and high background noise.
Following incubation, wash blots three times with TBST to remove unbound secondary antibody.
Protein Visualization
Use a substrate called BCIP/NBT which reacts with alkaline phosphatase to produce a visible signal.
Alkaline phosphatase (conjugated to the secondary antibody) facilitates the BCIP/NBT system.
Based on the hydrolysis of BCIP and reduction of NBT, producing a deep purple reaction product if the protein of interest is present. The intensity of the band correlates with the amount of protein present.
Results and Analysis
Two final blots are probed with total MAP kinase (left) and active MAP kinase antibody (right).
Total MAP kinase blot shows the protein marker, control PC12 cell extract, and stimulated PC12 cell extract.
Both control and stimulated samples contain total MAP kinase at equivalent levels. This indicates that the total amount of MAP kinase is constant.
Total MAP kinase detection includes both activated and non-activated forms.
Active MAP kinase blot shows the protein marker, control PC12 cell extract, and stimulated PC12 cell extract.
The control sample does not contain activated MAP kinase, while the stimulated sample does. This shows that NGF stimulation is required for MAP kinase activation.
Conclusion: Treating PC12 cells with NGF results in the activation/phosphorylation of MAP kinase.
Two distinct bands for each sample represent different isoforms of MAP kinase. These isoforms may have slightly different functions or regulatory properties.
Top band: 44 kDa isoform.
Lower band: 42 kDa isoform.
Protein isoforms are proteins similar to each other with similar roles within cells.
A single gene can encode multiple isoforms via alternative splicing (e.g., MAP kinase). Alternative splicing allows for increased protein diversity from a single gene.