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SDS-PAGE &
Western Blot

Master the techniques of protein separation by size and specific protein detection using gel electrophoresis and antibody-based identification.

Biochemistry

What is SDS-PAGE?

SDS-PAGE stands for Sodium Dodecyl Sulfate: Polyacrylamide Gel Electrophoresis. It is a technique used to separate proteins based solely on their molecular weight.

SDS
An anionic detergent that denatures proteins and coats them with a uniform negative charge proportional to mass.
PAGE
A porous polyacrylamide gel matrix that acts as a molecular sieve, separating proteins as they migrate through an electric field.

How SDS Denatures Proteins

Native Protein Folded + - + + SDS + Heat Denatured Protein - - - - - - - - - Uniform negative charge Reducing agent (e.g., BME): Breaks disulfide bonds (S-S) S-S broken

The Polyacrylamide Gel Matrix

The gel acts as a molecular sieve. The percentage of acrylamide determines the pore size, which controls the resolution range for different protein sizes.

Low % Gel (\(6\)-\(8\)%)
Large pores: best for resolving high molecular weight proteins (\(100\)-\(250\) kDa)
Medium % Gel (\(10\)-\(12\)%)
Medium pores: resolves proteins in the mid-range (\(20\)-\(100\) kDa)
High % Gel (\(15\)%)
Small pores: best for resolving small proteins (<\(20\) kDa)

How Proteins Separate in the Gel

Stacking Gel (\(4\)%) M 1 2 3 4 Resolving Gel (\(12\)%) 250 130 70 35 25 10 kDa (-) (+) Migration Large (slow) Small (fast)

Mobility vs. Molecular Weight

In SDS-PAGE, the relative mobility (\( R_f \)) of a protein is inversely proportional to the logarithm of its molecular weight:

Relative Mobility (\(R_f\))
\(\log(MW)\)
 250 kDa 130 kDa 70 kDa 35 kDa 10 kDa Unknown

Plot the known standards to create a standard curve. Then read off the unknown protein's molecular weight from its \( R_f \) value.

What is a Western Blot?

A Western blot (also called an immunoblot) is a technique that uses antibodies to detect a specific protein after it has been separated by SDS-PAGE. It answers the question: "Is my protein of interest present, and at what size?"

Specificity
Antibodies bind only the target protein, even among thousands of others
Sensitivity
Can detect proteins at nanogram or even picogram levels
Size Info
Confirms the molecular weight of the detected protein

Western Blot Workflow

1
SDS-PAGE: Separate proteins by molecular weight in a polyacrylamide gel.
2
Transfer: Electroblot proteins from the gel onto a membrane (PVDF or nitrocellulose).
3
Blocking: Incubate membrane with a blocking agent (e.g., \(5\)% milk or BSA) in TBST buffer for \(1\) hour.
4
Primary Antibody: Add the primary antibody that specifically binds your target protein.
5
Secondary Antibody: Add a labeled secondary antibody that binds the primary antibody, enabling detection.
6
Detection: Visualize bands using chemiluminescence, fluorescence, or colorimetric methods.

Transfer & Antibody Detection

Gel Transfer Membrane
\(1^\circ\) Ab
+ Primary Ab
\(1^\circ\) Ab
\(2^\circ\) Ab
 ★ Signal
Sandwich: Protein → Primary Ab → Secondary Ab (labeled)
Secondary Ab carries enzyme/fluorophore for detection

SDS-PAGE vs. Western Blot

SDS-PAGE Western Blot
Purpose Separate proteins by size Identify a specific protein
Detection Non-specific stain (Coomassie, silver) Antibody-based (specific)
Information Protein profile: number, size, abundance Presence, size, and relative amount of target
Sensitivity Moderate (ng range) High (pg to ng range)
Requires antibodies? No Yes
Typical use Purity check, total protein view Confirm expression of specific protein

How to Run a Western Blot

1
Prepare samples: Lyse cells, add SDS sample buffer with reducing agent, and boil for \(5\) min to denature proteins.
2
Load and run gel: Load samples and a molecular weight ladder into wells. Apply voltage (~\(100\)-\(200\) V) until the dye front reaches the bottom.
3
Transfer proteins: Electroblot from gel to PVDF or nitrocellulose membrane using a transfer buffer.
4
Block: Incubate membrane in \(5\)% non-fat milk or BSA in TBST buffer for \(1\) hour.
5
Probe with antibodies: Incubate with primary Ab (overnight at \(4\) °C), wash, then secondary Ab (\(1\) hr at RT), wash again.
6
Detect signal: Add substrate (e.g., ECL for chemiluminescence) and image the membrane.

Example: Reading an SDS-PAGE Gel

M Lysate Purified Control 150 100 75 50 25 No bands
~\(75\) kDa
 Target protein

Example: Interpreting a Western Blot

Western Blot Membrane M WT KO OE 100 75 50 37 ---
Loading control (actin, ~\(42\) kDa):
 Equal loading confirmed: all actin bands similar intensity

Example: Troubleshooting a Blot

A student runs a Western blot for a \(60\) kDa target protein but sees a band at ~\(120\) kDa and no band at \(60\) kDa. What happened?

Problem
Band at ~\(120\) kDa (double the expected size) suggests the protein is running as a dimer instead of a monomer.
Likely Cause
Insufficient denaturation: the sample was not boiled, or the reducing agent was omitted, leaving disulfide bonds intact.
Solution
Re-prepare the sample with fresh BME or DTT and boil for \(5\) min before re-running.

Common Mistakes

✕ Skipping the Blocking Step
Antibodies bind non-specifically across the entire membrane, producing a uniformly dark background with no distinct bands.
✕ Wrong Gel Percentage
Using \(15\)% gel for a \(200\) kDa protein: the protein barely enters the gel. Always match gel percentage to the target size range.
✕ Forgetting Loading Control
Without a loading control (like actin or GAPDH), you cannot tell if band intensity differences are due to protein levels or unequal loading.
✕ Mismatched Antibody Species
Using an anti-mouse secondary antibody when the primary was raised in rabbit: no signal because the secondary cannot bind the primary.

Summary Table

Concept Key Detail Why It Matters
SDS Denatures + adds uniform charge Separation by MW only
Reducing agent Breaks S-S bonds Prevents dimers/oligomers
Gel % Controls pore size Resolution of target MW range
Transfer Gel to membrane Makes proteins accessible to antibodies
Blocking Milk or BSA Prevents non-specific Ab binding
Primary Ab Binds target protein Specificity of detection
Secondary Ab Binds primary + carries label Signal amplification
Loading control Housekeeping protein (actin) Normalizes for equal loading

Question 1 of 5

What is the primary role of SDS in SDS-PAGE?

Question 2 of 5

In a properly run SDS-PAGE gel, which protein will migrate the farthest from the wells?

Question 3 of 5

Why is a blocking step essential in a Western blot?

Question 4 of 5

A researcher probes a Western blot for a \(50\) kDa protein and sees a strong band at \(100\) kDa but nothing at \(50\) kDa. What is the most likely explanation?

Question 5 of 5

What is the function of the secondary antibody in a Western blot?