Description
PVDF membrane for Western blot
GenieBlot PVDF Membrane (0.45 μm) is a high-binding PVDF transfer membrane for Western blotting of standard and high-molecular-weight proteins. It offers high binding capacity, mechanical strength and low background. Brief methanol activation is needed before transfer.
The hydrophobic polyvinylidene fluoride membrane has a uniformly controlled pore structure with a high binding capacity for biomolecules. It transfers proteins from a range of gel matrices, with higher adsorption for proteins >20 kDa, and detects total protein down to the nanogram level. Membranes can be stripped and re-probed with clear bands. Beyond Western blotting, the membrane suits amino acid analysis, glycoprotein staining, lipopolysaccharide analysis, N-terminal protein sequencing, dot blots and slot blots. For research use only.
Key Features
| High transfer efficiency | Strong protein adsorption detects total protein down to the nanogram level. | |
| Strip and re-probe | Membranes regenerate well after stripping and still give clear bands. | |
| Uniform pore structure | Controlled 0.45 μm pores give a high binding capacity for biomolecules. | |
| Versatile | Suits Western, dot and slot blots, N-terminal sequencing and more. |
Specifications
| Material | Hydrophobic polyvinylidene fluoride (PVDF) |
| Pore size | 0.45 μm |
| Recommended for | Proteins >20 kDa |
| Applications | Western blot, amino acid analysis, glycoprotein staining, lipopolysaccharide analysis, N-terminal protein sequencing, dot and slot blots |
| Activation | Anhydrous methanol, 30 sec - 1 min |
| Shelf life | 2 years |
Components
| Components | Amount |
| GenieBlot PVDF Membrane (0.45 μm) | 27.5 cm × 3.75 m / 90 mm × 60 mm, 50 sheets |
Storage
Store at 15 ~ 25°C.
Buffers
Optimise buffer concentrations for your experiment; a pilot experiment is recommended.
- Transfer buffer: 25 mM Tris base, 192 mM glycine, 20% methanol.
- Wash buffer: TBS or PBS with 0.1% Tween-20.
- TBS: 18.5 mM Tris base, pH 7.5, 0.9% NaCl.
- PBS: 10 mM phosphate, pH 7.4, 0.9% NaCl.
- Blocking buffer: 0.5%–5% (w/v) BSA, casein or non-fat milk powder in wash buffer.
| Blocking agent | Application | Concentration |
| Bovine serum albumin (BSA) | Commonly used for Western blot blocking | 0.5%–5% (w/v) |
| Casein / non-fat milk powder | Routine protein blocking; not suitable for phosphorylated proteins | 0.5%–5% (w/v) |
- Transfer the proteins
- After electrophoresis, equilibrate the gel in transfer buffer for 10–15 min.
- Activate the membrane in methanol for 30 sec - 1 min, until it turns evenly from white to semi-transparent grey.
- Equilibrate the membrane in transfer buffer for 30 sec - 1 min so residual methanol does not affect transfer.
- Handle the membrane with tweezers to avoid damage and contamination, and do not let dry spots form.
- Assemble the transfer stack (or follow your device's instructions), in order: gel clamp, filter paper, PVDF membrane, polyacrylamide gel, filter paper, gel clamp. Proteins transfer from the gel towards the membrane.
- Immunodetection
- Rinse the membrane briefly in wash buffer, then block on a shaker for 1 h at room temperature.
- Incubate with primary antibody (diluted in wash or blocking buffer) on a shaker for 2 h at room temperature or overnight at 4°C.
- Wash 3–5 times for 5 min each.
- Incubate with secondary antibody (diluted in wash or blocking buffer) on a shaker for 1 h at room temperature.
- Wash 3–5 times for 10 min each.
- Drain the membrane on filter paper, cover it evenly with ECL working solution and incubate for 30 sec - 1 min at room temperature. Expose to X-ray film or image with a CCD imager.
- Choose the ECL substrate according to target abundance: GenieBlot Femto ECL Substrate Kit for low-abundance targets, or GenieBlot Pico ECL Substrate Kit for routine targets.
Q1: Does the membrane need activating before use?
- Yes. Soak it in anhydrous methanol for 30 sec - 1 min until it turns evenly semi-transparent grey, then equilibrate it in transfer buffer for 30 sec - 1 min.
Q2: What protein sizes is the 0.45 μm membrane best for?
- It adsorbs proteins with a molecular weight >20 kDa more efficiently.
Q3: Can the membrane be stripped and re-probed?
- Yes. The membrane regenerates well after stripping and gives clear bands.
Q4: Which blocking agent should I use for phosphorylated proteins?
- Use BSA. Casein and non-fat milk powder are suitable for routine proteins but not for phosphorylated proteins.
