Western Blot Hub

Western Blot Wet Transfer: Step-by-Step Protocol

Wet transfer is the most commonly used method for western blot protein transfer. It provides excellent transfer efficiency for proteins of all sizes and is especially recommended for large proteins (>100 kDa). This detailed protocol covers every step from gel preparation to transfer verification.

Overview

Wet transfer involves submerging the gel-membrane sandwich in a tank filled with transfer buffer. An electric field drives proteins from the gel toward the membrane. This method is:

  • Most reliable: Excellent transfer efficiency for all protein sizes
  • Best for large proteins: Superior to semi-dry for proteins >100 kDa
  • Versatile: Works well for most applications
  • Requires cooling: Must be run at 4°C or with cooling system
  • Longer time: Typically 1-3 hours (or overnight)

The key to successful wet transfer is proper assembly of the transfer stack, complete removal of air bubbles, and maintaining cold temperature throughout the transfer.

Materials Required

Reagents

  • • Transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol)
  • • PVDF or nitrocellulose membrane
  • • 100% methanol (for PVDF activation)
  • • Ponceau S stain (for verification)

Equipment

  • • Transfer buffer tank
  • • Transfer cassette
  • • Filter paper (Whatman 3MM or equivalent)
  • • Transfer sponges or pads
  • • Cooling system or ice
  • • Power supply
  • • Test tube or roller (for removing bubbles)

Step-by-Step Procedure

Step 1: Gel Equilibration

After electrophoresis, carefully remove the gel from the glass plates. Handle the gel gently to avoid tearing.

Equilibrate the gel in transfer buffer for 15-30 minutes with gentle shaking. This step:

  • Removes excess SDS from the gel
  • Improves transfer efficiency
  • Prevents gel swelling during transfer
  • Is especially important for large proteins

Important: Do not skip gel equilibration - it significantly improves transfer efficiency, especially for large proteins.

Step 2: Membrane Preparation

Cut the membrane to size (slightly larger than the gel) using clean scissors or a scalpel.

For PVDF Membrane:

  1. Activate membrane by immersing in 100% methanol for 30 seconds
  2. Transfer to transfer buffer immediately
  3. Equilibrate in transfer buffer for 5 minutes
  4. Keep membrane wet throughout the process

For Nitrocellulose Membrane:

  1. No methanol activation needed
  2. Wet directly in transfer buffer
  3. Equilibrate for 5 minutes
  4. Handle carefully as it's more fragile

Step 3: Transfer Stack Assembly

Assemble the transfer stack in a tray filled with transfer buffer. This prevents the stack from drying out and makes bubble removal easier.

Stack order (from cathode/black/negative to anode/red/positive):

  1. Sponge or pad (cathode side)
  2. 3 pieces of filter paper (soaked in transfer buffer)
  3. Gel (face down, protein side toward membrane)
  4. Membrane (on top of gel, protein-binding side toward gel)
  5. 3 pieces of filter paper (soaked in transfer buffer)
  6. Sponge or pad (anode side)

Critical step - Remove ALL air bubbles:

  • Roll each layer with a test tube or roller
  • Start from the center and roll outward
  • Check for bubbles between gel and membrane (most critical)
  • Air bubbles prevent protein transfer at that location

Warning: Even small air bubbles can cause complete transfer failure at that spot. Take time to remove all bubbles thoroughly.

Step 4: Close Cassette and Place in Tank

Once the stack is assembled and all bubbles are removed:

  1. Close the transfer cassette carefully
  2. Ensure the cassette is oriented correctly (cathode toward cathode, anode toward anode)
  3. Place cassette in transfer tank filled with cold transfer buffer
  4. Ensure buffer completely covers the cassette
  5. Add ice or use cooling system to maintain 4°C

The cassette should be completely submerged, and the buffer should be well-stirred during transfer (if using a stir bar).

Step 5: Transfer Verification

After transfer is complete, verify successful transfer before proceeding:

  1. Carefully remove membrane from cassette
  2. Stain with 0.1% Ponceau S in 5% acetic acid for 2-5 minutes
  3. Rinse with water to visualize protein bands
  4. Document or photograph if needed
  5. Wash with TBST before blocking (stain will be removed)

Ponceau S staining confirms transfer success and shows transfer quality. All expected bands should be visible.

Transfer Conditions

Transfer conditions depend on protein size and your time constraints:

Standard Conditions

  • Voltage: 100V constant voltage
  • Time: 1 hour
  • Temperature: 4°C (with ice or cooling system)
  • Best for: Most proteins, standard applications

Large Proteins (>100 kDa)

  • Voltage: 70-80V constant voltage
  • Time: 2-3 hours
  • Temperature: 4°C
  • Buffer modification: Reduce methanol to 10%, add 0.1% SDS
  • Best for: High molecular weight proteins

Overnight Transfer

  • Voltage: 30V constant voltage
  • Time: Overnight (12-16 hours)
  • Temperature: 4°C
  • Best for: When time allows, very gentle transfer
  • Advantages: Gentle, efficient, no need to monitor

Transfer Buffer Recipe

Standard transfer buffer (1L):

  • 25 mM Tris base (3.03 g)
  • 192 mM glycine (14.4 g)
  • 20% methanol (200 mL) - for PVDF
  • 10% methanol (100 mL) - for large proteins or nitrocellulose
  • 0.1% SDS (1 g) - optional, for large proteins
  • Add water to 1L, pH should be ~8.3

Optimization Tips

For Better Transfer Efficiency

  • Always equilibrate gel in transfer buffer (15-30 min)
  • Remove ALL air bubbles thoroughly
  • Use fresh transfer buffer for each transfer
  • Maintain cold temperature (4°C) throughout
  • Ensure adequate buffer volume in tank

For Large Proteins

  • Reduce methanol to 10% (improves large protein transfer)
  • Add 0.1% SDS to transfer buffer
  • Use lower voltage (70-80V) for longer time (2-3 hours)
  • Extend gel equilibration time (30 minutes)
  • Consider overnight transfer at 30V

Troubleshooting

Incomplete Transfer

Solutions:

  • Extend transfer time (especially for large proteins)
  • Reduce methanol to 10%
  • Add 0.1% SDS to transfer buffer
  • Check voltage and current settings
  • Verify buffer pH and freshness

Bubbles on Membrane

Solutions:

  • Ensure no air bubbles during assembly
  • Roll thoroughly with test tube or roller
  • Work in a tray filled with buffer
  • Check for bubbles before closing cassette

Uneven Transfer

Solutions:

  • Check buffer levels in transfer tank
  • Ensure good contact between gel and membrane
  • Verify power supply is working correctly
  • Check cassette assembly and orientation

Gel Sticking to Membrane

Solutions:

  • Ensure gel is properly equilibrated
  • Use fresh transfer buffer
  • Check buffer composition (correct methanol percentage)
  • Verify transfer conditions

Related Protocols

Protein Transfer Overview

Complete guide to protein transfer methods

Semi-Dry Transfer Protocol

Guide to semi-dry transfer method

Transfer Optimization

Tips for optimizing transfer conditions

Complete Western Blot Protocol

Full step-by-step western blot guide