Description
PVDF membrane for small proteins and peptides
GenieBlot PVDF Membrane (0.22 μm) is a high-binding PVDF transfer membrane optimised for low-molecular-weight proteins and peptides. It provides efficient retention, high binding capacity and low background, and needs only brief alcohol activation before transfer.
The hydrophobic polyvinylidene fluoride membrane has a uniformly controlled 0.22 μm pore structure with high binding capacity for biomolecules, and gives higher adsorption of proteins smaller than 20 kDa. It is mainly used for Western blotting from a range of gel matrices, with a total-protein detection threshold at the nanogram level. It also suits amino acid analysis, glycoprotein staining, lipopolysaccharide analysis, N-terminal protein sequencing, dot blots and slot blots. Membranes strip and reprobe well, giving clear bands. For research use only.
Key Features
| High transfer efficiency | Strong protein adsorption gives nanogram-level total protein detection. | |
| Optimised for small proteins | The 0.22 μm pore size gives higher adsorption of proteins smaller than 20 kDa. | |
| Strip and reprobe | Membranes regenerate well after stripping and still give clear bands. | |
| Versatile | Suits Western, dot and slot blots, N-terminal sequencing and other membrane-based analyses. |
Specifications
| Material | Hydrophobic polyvinylidene fluoride (PVDF) |
| Pore size | 0.22 μm |
| Optimal targets | Proteins and peptides <20 kDa |
| Applications | Western blot, amino acid analysis, glycoprotein staining, lipopolysaccharide analysis, N-terminal sequencing, dot blot, slot blot |
| Activation | Anhydrous methanol, 30 sec - 1 min |
| Shelf life | 2 years |
Components
| Components | Amount |
| GenieBlot PVDF Membrane (0.22 μm) | 27.5 cm × 3.75 m / 90 mm × 60 mm, 50 sheets |
Storage
Store at 15 ~ 25°C.
Buffers
Determine 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 containing 0.1% Tween-20.
- TBS: 18.5 mM Tris base, pH 7.5, 0.9% NaCl.
- PBS: 10 mM phosphate salt, 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 | Usage |
| Bovine serum albumin (BSA) | Commonly used for blocking in Western blot | 0.5%–5% (W/V) |
| Casein / non-fat milk powder | Blocking for routine proteins in Western blot; 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 PVDF membrane in methanol for 30 sec - 1 min. It turns evenly from white to semi-transparent grey.
- Equilibrate the membrane in transfer buffer for 30 sec - 1 min so that residual methanol does not affect transfer.
- Handle the membrane with tweezers to avoid damage and contamination, and do not leave dry spots, which inhibit transfer.
- Assemble the transfer stack as per your transfer device instructions, in this order: gel clamp, filter paper, PVDF membrane, polyacrylamide gel, filter paper, gel clamp.
- Immunodetection (optimise as required)
- Rinse the membrane briefly in wash buffer, then block in blocking buffer on a shaker for 1 h at room temperature. Dilute the primary antibody in wash or blocking buffer.
- Incubate the membrane in primary antibody on a shaker for 2 h at room temperature or overnight at 4°C.
- Wash 3–5 times for 5 min each with wash buffer. Dilute the secondary antibody in wash or blocking buffer.
- Incubate in secondary antibody on a shaker for 1 h at room temperature.
- Wash 3–5 times for 10 min each with wash buffer.
- Drain the membrane on filter paper, cover evenly with ECL working solution and incubate at room temperature for 30 sec - 1 min. Expose to X-ray film or image on a CCD imager.
- Choose the ECL kit according to target abundance: the GenieBlot Femto ECL Substrate Kit for low-abundance proteins or the GenieBlot Pico ECL Substrate Kit for conventional targets.
Q1: Does the membrane need activation before use?
- Yes. Pre-treat it in anhydrous methanol for 30 sec - 1 min until it turns evenly from white to semi-transparent grey, then equilibrate in transfer buffer for 30 sec - 1 min.
Q2: When should I choose the 0.22 μm pore size?
- The 0.22 μm membrane gives higher adsorption of proteins smaller than 20 kDa, so it is the better choice for low-molecular-weight proteins and peptides.
Q3: Can I strip and reprobe the membrane?
- 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.
