WB 0.45μm PVDF Membrane 750px,TR50045

Product Introduction

This Western Blot (WB) 0.45μm PVDF (Polyvinylidene Fluoride) Membrane is a high-quality microporous membrane designed for the efficient transfer and immobilization of proteins in western blotting. Supplied as a 750px wide roll, it offers flexibility for cutting to custom sizes, making it suitable for various gel formats and high-throughput applications.

The 0.45μm pore size is the standard choice for routine protein analysis, providing an optimal balance between protein retention and transfer speed. This membrane is characterized by its robust physical strength, high protein binding capacity, and excellent compatibility with all common detection methods.

Product Features

Ideal for Standard & Large Proteins: The 0.45μm pore size is perfectly suited for the efficient transfer and immobilization of proteins with molecular weights greater than 20 kDa.

   High Protein Binding Capacity: Offers strong protein affinity (typically >150 µg/cm²), ensuring reliable detection for a wide range of targets.

   Superior Mechanical Durability: High tensile strength resists tearing and damage, allowing for easy handling and multiple rounds of stripping and reprobing.

   Excellent Chemical Resistance: Stable in methanol, ethanol, acetonitrile, and other common solvents used in blotting protocols.

   Versatile Detection Compatibility: Delivers low background and high signal-to-noise ratio for chemiluminescent, colorimetric, and fluorescent detection systems.

   Convenient Roll Format: The 750px width allows users to economically cut the membrane to the precise size required for their specific gel apparatus.

Usage Method (For Reference Only)

Important: Always handle the membrane with gloves or clean forceps to avoid contamination from skin proteins and oils.

A. Membrane Preparation (Pre-wetting):

1.  Cut the membrane to the size of your gel.

2.  Activate the membrane by immersing it in 100% methanol for 15-30 seconds. This critical step hydrates the hydrophobic PVDF, allowing transfer buffer to permeate the pores.

3.  Rinse the membrane briefly with deionized water to remove excess methanol.

4.  Equilibrate the membrane in cold transfer buffer for at least 5-10 minutes prior to assembling the transfer stack.

B. Western Blot Transfer (Standard Wet Transfer):

1.  Assemble the transfer stack in the following order (from cathode to anode):

       Cathode (-)

       Sponge / Pad

       Filter Paper

       SDS-PAGE Gel

       Activated PVDF Membrane

       Filter Paper

       Sponge / Pad

       Anode (+)

2.  Carefully roll a glass tube or pipette over the stack to remove all air bubbles, ensuring uniform contact between the gel and membrane.

3.  Perform electrophoresis transfer according to your standard protocol (e.g., 100V for 60-90 minutes at 4°C).

C. Post-Transfer Processing:

1.  Blocking: Incubate the membrane in a suitable blocking solution (e.g., 5% non-fat milk in TBST) for 1 hour at room temperature with gentle agitation.

2.  Antibody Incubations:

       Incubate with primary antibody (diluted in blocking buffer or TBST) for 1 hour at RT or overnight at 4°C.

       Wash 3x for 5-10 minutes each with TBST.

       Incubate with enzyme-conjugated secondary antibody (diluted in buffer) for 1 hour at RT.

       Wash 3x for 5-10 minutes each with TBST.

3.  Detection: Develop the blot using your preferred chemiluminescent, fluorescent, or colorimetric substrate.

Precautions

Glove Use: Always handle the membrane with gloves or forceps. Skin contact will deposit proteins and lipids, leading to high background.

   Mandatory Methanol Activation: Pre-wetting with methanol is absolutely required to make the PVDF membrane hydrophilic and functional. Using water or transfer buffer alone will not work.

   Bubble Removal: Meticulously remove all air bubbles during transfer stack assembly to prevent blank areas on the final blot.

   Storage Conditions: Store the roll in a cool, dry place, away from direct sunlight and chemical vapors.

   Chemical Safety: Methanol is hazardous. Handle it in a well-ventilated area while wearing appropriate personal protective equipment (PPE).

Frequently Asked Questions (FAQ)

Q1: When should I choose a 0.45μm membrane over a 0.22μm membrane?

A: The 0.45μm membrane is the ideal standard for most proteins larger than 20 kDa. It provides faster transfer times. Choose the 0.22μm membrane for smaller proteins (<20 kDa, like peptides) to prevent them from passing through the larger pores.

Q2: Can I skip the methanol step and use only water or transfer buffer?

A: No. PVDF is hydrophobic. Methanol is necessary to wet the membrane and open the pores for aqueous buffers and proteins. Skipping this step will result in failed or very inefficient protein transfer.

Q3: Is this membrane suitable for stripping and reprobing?

A: Yes. The high mechanical strength of PVDF makes it highly durable and well-suited for multiple rounds of stripping and reprobing with standard stripping buffers.

Q4: I see uneven staining or blank spots on my blot. What happened?

A: This is most commonly caused by:

   Air Bubbles: Trapped between the gel and membrane during transfer assembly.

   Improper Activation: Incomplete or uneven wetting of the membrane with methanol.

   Contamination: From gloves or dirty surfaces.

Q5: Can I use this membrane for fluorescent western blotting?

A: Yes, it is compatible. However, standard PVDF can have some inherent autofluorescence, which may be noticeable with highly sensitive fluorescent detectors. For optimal performance in fluorescent applications, a specially formulated low-fluorescence PVDF membrane is recommended.

Q6: Why is my background signal too high?

A: High background is typically related to the immunodetection steps, not the membrane itself. Consider:

   Optimizing your blocking conditions (time, concentration, and type of blocker).

   Titrating your primary and secondary antibody concentrations.

   Increasing the number and duration of washes.

   Ensuring the membrane does not dry out during the process.

Product Disclaimer

This product is intended for research use only. If intended for the storage of valuable samples, please conduct a small-scale pilot experiment prior to large-scale use. The product warranty is strictly limited to the product itself and does not cover any other incidental or consequential damages. Please be advised.

This product is for research purposes only. It is not intended for use in clinical diagnosis or therapy.