Immunoblotting, also known as Western Blot, is a hybridization technique that combines high-resolution gel electrophoresis with immunochemical analysis. The key to immunoblotting is immobilizing the target proteins on a solid support (membrane) for subsequent detection.
Polyvinylidene fluoride (PVDF) membrane is one of the typical immunoblotting membranes, suitable for binding various proteins. PVDF membrane is naturally hydrophobic with a uniform pore structure, offering strong protein-binding capacity. Aladdin's transfer membranes are hydrophobic and available in two pore sizes: 0.45 μm and 0.22 μm. The 0.45 μm pore size is suitable for proteins of most molecular weights, while the 0.22 μm pore size is more appropriate for proteins smaller than 20 kDa.
Aladdin PVDF membranes exhibit high mechanical strength, resist irregular breakage during cutting, and are compatible with both chemiluminescent and fluorescent detection methods. Aladdin PVDF membranes are not sided; either side can face the gel during use.
This manual provides a reference wet transfer protocol. Optimal transfer conditions may require adjustment for different proteins.
Product Code
| Pore Size
| Size
| Format
|
P1492692-A01
| 0.22μm
| 27.5cm×3.75m
| Roll
|
P1492692-A02
| 0.22μm
| 8.4×7cm
| Sheet
|
P1492692-A03
| 0.22μm
| 15×15cm
| Sheet
|
P1492692-A04
| 0.22μm
| 20×20cm
| Sheet
|
P1492692-B01
| 0.45μm
| 27.5cm×3.75m
| Roll
|
P1492692-B02
| 0.45μm
| 8.4×7cm
| Sheet
|
P1492692-B03
| 0.45μm
| 15×15cm
| Sheet
|
P1492692-B04
| 0.45μm
| 20×20cm
| Sheet
|
Recommended Materials for Western Blotting
Reagent
| Preparation Method
|
30% (W/V) Acrylamide Monomer Solution
| 29g Acrylamide, 1g Bis-acrylamide. Add deionized water to 100mL. Adjust pH to ≤7.0. Store in a brown bottle at 4°C.
|
10% Ammonium Persulfate (APS) (-20°C storage)
| 1g APS. Add deionized water to 10mL. Aliquot into 10 tubes, 1mL each.
|
1.5M Tris-HCl (pH 8.8)
| 18.2g Tris base. Adjust pH to 6.8 with HCl, then add deionized water to 100mL. Readjust pH to 8.8.
|
1M Tris-HCl (pH 6.8)
| 12.12g Tris base. Adjust pH to 6.8 with HCl, then add deionized water to 100mL. Readjust pH to 6.8.
|
10x SDS-PAGE Running Buffer
| 30g Tris base, 144g Glycine, 10g SDS. Add deionized water to 1000mL. pH ~8.3.
|
6x SDS Loading Buffer
| 0.5M Tris pH6.8 (7.0mL), SDS (1.0g, 0.35M final), Glycerol (3.0mL, 30% V/V), DTT (0.93g, 0.6M final), Bromophenol Blue (1.2mg, 0.175mM final).
|
Transfer Buffer
| 25mM Tris, 192mM Glycine, 20% (V/V) Methanol. Dissolve 3.03g Tris and 14.4g Glycine in 500mL deionized water. Add 200mL methanol, bring to 1L with water.
|
TBS Buffer
| 20mM Tris-HCl, 500mM NaCl. Dissolve 4.84g Tris and 58.48g NaCl in 1.5L water. Adjust pH to 7.5 with HCl. Bring to 2L with deionized water.
|
TBST Wash Buffer
| 20mM Tris-HCl, 500mM NaCl, 0.1% Tween 20. Add 1mL Tween 20 to 1L of TBS buffer.
|
Blocking Buffer
| 5% Skim Milk in TBS. Dissolve 5.0g skim milk powder in 100mL TBS buffer.
|
Antibody Dilution Buffer
| 5% Skim Milk in TBST. Dissolve 5.0g skim milk powder in 100mL TBST buffer.
|
Primary Antibody (target specific)
| Use according to the supplier's instructions. Typically diluted in Antibody Dilution Buffer.
|
Secondary Antibody (HRP conjugated, specific to primary antibody host)
| Use according to the supplier's instructions. Typically diluted in Antibody Dilution Buffer.
|
Other Materials
| PVDF membrane, Filter paper, Sponge pads, Pure water, 100% Methanol |
User Notes
1.Aladdin transfer membranes are hydrophobic and must be activated in absolute methanol or absolute ethanol prior to use. To activate, gently immerse the cut membrane in a container of absolute methanol/ethanol using forceps, ensuring it is fully submerged. Soak for at least 1 minute to ensure complete activation. Successful activation is indicated by the membrane turning from opaque white to semi-transparent. Subsequently, transfer the membrane to transfer buffer for equilibration.
2.During equilibration, the membrane may float on the surface due to altered hydrophobicity. Gently submerge it into the transfer buffer using forceps to ensure complete replacement of the methanol/ethanol with transfer buffer. Soak for at least 1 minute.
3.Once activated, the membrane must be kept wet throughout the subsequent transfer and antibody incubation steps. Allowing the membrane to dry will directly affect the final band pattern, potentially causing missing bands, high background, or other anomalies.
Protocol
1. SDS-PAGE Electrophoresis
1.1 Align the glass plates and secure them vertically in the casting frame.
Note: Ensure the plates are perfectly aligned to prevent leakage.
1.2 Prepare the separating gel solution according to the desired concentration (see table below). Add TEMED, mix immediately, and pour the gel along the space between the plates until the level is about 2 cm from the top. Carefully overlay the gel with water or butanol for sealing.
Note: Pour the gel carefully to avoid bubbles. Overlay slowly to prevent gel distortion.
1.3 Once a distinct interface appears between the gel and the overlay (indicating polymerization), wait an additional 5 minutes for complete solidification. Pour off the overlay and blot away any residual liquid with filter paper.
1.4 Prepare the 5% stacking gel solution (see table below). Add TEMED, mix immediately, and pour the remaining space. Insert the comb horizontally. After the stacking gel has polymerized, carefully remove the comb by pulling it straight up. The gel is now ready for electrophoresis.
Separating Gel Formulations (for 6 mL total volume)
| Reagent | 6% | 8%
| 10%
| 12%
| 15%
|
H₂O (mL)
| 3.18 | 2.78 | 2.38 | 1.98 | 1.38 |
| 30% Acrylamide (29:1) (mL) | 1.2 | 1.6 | 2.0 | 2.4 | 3.0 |
| 1.5M Tris-HCl (pH8.8) (mL) | 1.5 | 1.5
| 1.5
| 1.5
| 1.5
|
10% SDS (μL)
| 60 | 60
| 60
| 60
| 60
|
10% APS (μL)
| 60
| 60
| 60
| 60
| 60
|
TEMED (μL)
| 3 | 3
| 3
| 3
| 3 |
Stacking Gel Formulation (5%, for 3 mL total volume)
| 试剂 Reagent | Volume |
H₂O (mL)
| 2.0 |
| 30% Acrylamide (29:1) (mL) | 0.5 |
1.5M Tris-HCl (pH6.8) (mL)
| 0.5
|
10% SDS (μL)
| 40 |
10% APS (μL)
| 30 |
TEMED (μL)
| 4 |
1.5 Measure the protein concentration of your samples. Calculate the volume containing the desired amount of protein (e.g., 1 μg, 0.5 μg) – this is the loading volume. Transfer this volume to a 0.5 mL tube and add the appropriate volume of 6x SDS Loading Buffer.
1.6 Denature the samples by heating at 95-100°C for 5 minutes.
1.7 Fill the electrophoresis chamber with running buffer, ensuring the inner chamber is filled above the level of the wells. Using a pipette, slowly load the samples into the wells, avoiding introducing bubbles.
1.8 Run the gel at constant voltage (e.g., 90V through the stacking gel, 150V through the separating gel) until the bromophenol blue dye front reaches about 1 cm from the bottom of the gel. Stop electrophoresis and proceed to transfer.
2. Wet Transfer
2.1 Carefully disassemble the gel unit and rinse the gel with pure water. Gently remove the stacking gel. Equilibrate the gel in transfer buffer for 5 min.
2.2 Activate the PVDF membrane by immersing it in 100% methanol for at least 1 min until it turns uniformly semi-transparent. Equilibrate the activated membrane in transfer buffer for at least 1 min.
2.3 Cut 6 pieces of filter paper slightly narrower than the gel (approx. 1mm less). Soak them in transfer buffer.
2.4 Place the transfer cassette, sponge pads, a glass rod, the soaked filter papers, and the activated membrane in a tray containing transfer buffer.
2.5 Open the cassette (black side down). Place a sponge pad on the black side and roll with a glass rod to remove bubbles. Place 3 sheets of filter paper on the pad and roll out bubbles. Carefully place the equilibrated gel on the filter paper, align, and roll out any bubbles. Place the activated membrane on top of the gel, covering it completely (Do not move the membrane once placed). Roll out bubbles. Place the remaining 3 sheets of filter paper on the membrane and remove bubbles. Top with the second sponge pad, roll gently, and close the cassette.
Note: Perform all steps submerged in transfer buffer to minimize bubble formation, which hinders transfer.
2.6 Place the cassette into the transfer tank ensuring the black side faces the black (cathode) side of the tank. Fill the tank with 1L of transfer buffer. Place the tank in an ice bath or cold water bath during transfer to prevent overheating. Transfer at constant current: 300 mA for 30-90 min.
Note: Using pre-stained protein markers is recommended to monitor transfer efficiency.
3. Immunodetection
The following is a general protocol. Refer to the antibody and detection reagent manufacturers' instructions for optimal results.
3.1 Briefly rinse the membrane with TBST to remove any residual gel. Place the membrane (protein side up) in a container with blocking buffer. Block by incubating on a rocking platform at room temperature for 1-2 hours (2 hours is optimal).
3.2 Discard the blocking buffer. Incubate the membrane with the primary antibody diluted in TBST (or Antibody Dilution Buffer) for 1-2 hours at room temperature (or overnight at 4°C) with gentle shaking. Discard or recover the primary antibody. Wash the membrane three times for 10 minutes each with TBST on a shaker at room temperature.
3.3 Incubate the membrane with the HRP-conjugated secondary antibody diluted in TBST (or Antibody Dilution Buffer) for 1-2 hours at room temperature with gentle shaking. Discard or recover the secondary antibody. Wash the membrane three times for 10 minutes each with TBST on a shaker.
3.4 Detection (Chemiluminescence or Chromogenic)
Perform detection using an appropriate method (e.g., ECL reagent) according to the manufacturer's instructions and image the blot.
Precautions
This product is for research use by professionals only. Use for clinical diagnosis, therapy, or in food or pharmaceutical applications has not been validated and should be approached with caution. For your safety and health, please wear a lab coat and disposable gloves during operation.
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