Western Blot 0.22μm PVDF Membrane 750px,TR50022

Product Introduction

This Western Blot (WB) 0.22μm PVDF (Polyvinylidene Fluoride) Membrane is a premium microporous membrane specifically engineered for the transfer and immobilization of proteins in western blotting applications. Supplied in a convenient 750px width roll, it is ideal for processing multiple samples or large proteins.

PVDF membrane is renowned for its high binding capacity, superior mechanical strength, and excellent chemical resistance. It is particularly effective for the detection of low-abundance proteins and is the membrane of choice for a wide range of applications, including routine protein analysis, phosphoprotein detection, and the blotting of large molecular weight proteins.

Product Features

High Protein Binding Capacity: Exhibits a high affinity for a broad range of proteins, typically >150 µg/cm², ensuring strong signal detection, especially for low-abundance targets.

   Optimal Pore Size (0.22μm): Ideal for trapping most proteins, particularly those with molecular weights below 100 kDa, providing sharp bands and high resolution.

   Exceptional Mechanical Strength: Highly durable and tear-resistant, allowing for easy handling, multiple stripping/reprobing cycles, and prolonged incubations without damage.

   Superior Chemical Resistance: Compatible with a wide range of solvents, buffers, and detection substrates, including methanol and acetonitrile.

   Low Background & High Signal-to-Noise Ratio: Provides a clean background for enhanced clarity and sensitivity in chemiluminescent, fluorescent, and colorimetric detection methods.

   Pre-cut Roll Format: The 750px width allows for flexible cutting to custom sizes to fit various gel and transfer apparatus dimensions.

Usage Method (For Reference Only)

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

A. Membrane Preparation (Pre-wetting):

1.  Cut the membrane to the desired size.

2.  Immerse the membrane briefly in 100% methanol for 15-30 seconds. This step is critical for hydrating and activating the PVDF surface, making it hydrophilic.

3.  Rinse the membrane thoroughly with deionized water for 1-2 minutes to remove residual methanol.

4.  Equilibrate the membrane in transfer buffer for at least 5 minutes before assembling the transfer stack.

B. Western Blot Transfer (Wet Transfer Method):

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

       Cathode (-)

       Fiber Pad / Sponge

       Filter Paper

       SDS-PAGE Gel

       Pre-wetted PVDF Membrane

       Filter Paper

       Fiber Pad / Sponge

       Anode (+)

2.  Ensure no air bubbles are trapped between the gel and the membrane.

3.  Perform electrophoretic transfer according to your standard protocol (e.g., 100V for 60 minutes or 25V overnight at 4°C).

C. Post-Transfer Processing:

1.  Blocking: Incubate the membrane in an appropriate blocking buffer (e.g., 5% non-fat dry milk or BSA in TBST) for 1 hour at room temperature with gentle agitation.

2.  Primary Antibody Incubation: Dilute the primary antibody in blocking buffer or antibody dilution buffer. Incubate with the membrane for 1 hour at room temperature or overnight at 4°C.

3.  Washing: Wash the membrane 3-4 times for 5-10 minutes each with TBST.

4.  Secondary Antibody Incubation: Incubate with the HRP (or other conjugate)-labeled secondary antibody diluted in buffer for 1 hour at room temperature.

5.  Washing: Repeat the washing step as above.

6.  Detection: Proceed with your chosen detection method (e.g., ECL substrate exposure to X-ray film or digital imager).

Precautions

Handle with Care: Always use clean forceps or wear powder-free gloves. Fingerprints can cause high background noise.

   Methanol Activation: Pre-wetting with 100% methanol is essential. Do not skip this step. Failure to do so will result in poor protein binding.

   Avoid Bubbles: Ensure perfect contact between the gel and membrane during transfer assembly to prevent blank spots on the blot.

   Storage: Store the unused membrane in its original packaging at room temperature, protected from light and moisture.

   Safety: Methanol is toxic and flammable. Please handle it in a well-ventilated area and wear appropriate personal protective equipment (PPE).

Frequently Asked Questions (FAQ)

Q1: Why must I activate the PVDF membrane with methanol?

A: PVDF is inherently hydrophobic. Methanol treatment displaces air from the pores and renders the surface temporarily hydrophilic, allowing aqueous buffers to penetrate and proteins to bind effectively.

Q2: What is the difference between 0.22μm and 0.45μm PVDF membranes?

A: The 0.22μm pore size is better for retaining smaller proteins (<20 kDa) and provides a higher binding capacity, reducing the risk of small proteins passing through the membrane. The 0.45μm pore size is often used for standard to large proteins (>20 kDa) and may offer slightly faster transfer times.

Q3: Can I re-probe (strip and re-use) this membrane?

A: Yes, the excellent mechanical strength of PVDF makes it suitable for stripping and reprobing. Common stripping buffers include mild glycine or commercial stripping solutions. However, some signal loss or background increase may occur with each cycle.

Q4: My background is high. What could be the cause?

A: High background can be caused by:

   Insufficient blocking.

   Contamination from hands or dirty equipment.

   Antibody concentrations that are too high.

   Inadequate washing. Ensure your blocking agent and antibody dilutions are optimized.

Q5: Why are there blank spots on my membrane after transfer?

A: This is almost always due to air bubbles trapped between the gel and the membrane during the assembly of the transfer stack. Carefully roll a glass rod or pipette over the stack to remove all bubbles.

Q6: Is this membrane suitable for fluorescent western blotting?

A: Yes, this PVDF membrane is compatible with fluorescent detection. However, it can exhibit some autofluorescence, particularly in the blue/green channels. It is recommended to check the background signal and consider low-fluorescence PVDF membranes for highly sensitive fluorescent applications.

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.