Western Transfer Using TotalBlot PVDF Membranes

Directions for use

 

TotalBlot PVDF membrane is a naturally hydrophobic polyvinylidene fluoride membrane which is well suited for Western protein transfers.  Immobilized proteins can be visually detected with all common staining reagents and can also be subjected directly to solid-phase protein sequencing and amino acid analysis.

A- Membrane Handling

TotalBlot PVDF membranes should be handled using gloves or forceps to prevent membrane contamination.  Either scissors or a sharp scalpel must be used to cut the membrane.  TotalBlot PVDF membranes must be wetted with 100% methanol before use.

 

B- Solutions

  • Sample buffer: 110 mM TrisHCl, pH 6.8; 16% (v/v) glycerol; 4% (w/v) SDS; 10% (v/v) 2-mercaptoethanol; 0.01% (w/v) Bromphenol Blue.

  • Transfer buffer: 25mM Tris base, 150mM glycine, 20% methanol, pH 8.3. 

  • 1X PBS: 150mM NaCl, 10mM NaPO4, pH 7.4.

  • BIO-BLOCK (Code E671/E667). Heat until dissolved. Other blocking agents may also be substituted (e.g. albumin, ethanolamine, gelatin, dried milk...), but optimal results will be obtained with BIO-BLOCK.

 

C- Gel Electrophoresis of Proteins

A standard protocol for SDS polyacrylamide gel electrophoresis is briefly described below.

  • Protein treatment:

  • Dilute protein sample to the range of 10 g/ml to 100 g/ml.

  • Add an equal volume of sample buffer (B.1.) to the protein samples. 

  • Heat protein solution at 100C for  5-10 min.

  • Electrophoresis:SDS polyacrylamide gel electrophoresis is performed in a 10% (or suitable concentration or gradient) polyacrylamide gel using the system developed by Laemmli1.

  • Load gel: Add 10-15 l of protein solution to each lane on the gel.

  • Apply an initial current of 25mA per gel and increase to 35mA per gel after the tracking dye migrates into the resolving gel.

  • Turn off the power supply when the run is completed.

 

D- Electrotransfer of Proteins from Gel to TotalBlot PVDF membranes.

Electroblotting conditions are determined by both the gel composition and biochemical state of the proteins being transferred.  Proteins denatured with SDS will have a net negative charge at neutral pH and will migrate to the anode under the influence of the applied field.  Electrophoretic movement of non-SDS denatured proteins (native) is dependent upon pH and ionic strength of the buffer.  For native proteins, the pH of the transfer buffer should be adjusted to give a net negative charge on the proteins of interest.  Under the optimal conditions, electrotransfers proceed in a relatively short time with high efficiency.

Transfer protocol:

  • Trim gel to area to be transferred.

  • Cut TotalBlot PVDF membrane the same size as the gel.

  • Wet TotalBlot PVDF membrane with 100% methanol for 15-30 sec. and then place membrane in transfer buffer. The membrane will now remain wet by aqueous solutions.

  • Assemble each gel section into a "sandwich" as follows:

  • Saturate filter paper with transfer buffer.

  • Place gel on one sheet of filter paper.

  • Place wetted TotalBlot PVDF membrane over gel. Eliminate bubbles by smoothing with gloved hand.

  • Place the other sheet of wet filter paper over the membrane.

  • Complete the sandwich by placing the gel and membranes between two Scotch Brite pads.

  • Fill electrotransfer apparatus with transfer buffer.

  • Insert assembly with gels and membranes into transfer apparatus with membrane  positioned between the gel and anode.

  • Apply 100 V for 2 to 4 hours.

 

E- Detection of Electrophoretically Transferred Proteins

After electrotransfer unlabeled proteins may be detected by several methods.  For direct protein staining, Coomassie Blue, Amido Black or Ponceau S may be used.

For total protein staining we recommend the following:

Solutions:

A. Stain Solution

B. Destain Solution

45 ml Methanol

2 ml Acetic Acid

53 ml distilled water

0.1 g Amido Black

180 ml methanol

4 ml acetic acid

16 ml distilled water

Protocol:

  • When transfer is complete add 50 ml stain solution to tray, then quickly transfer membrane to stain solution. Do not allow membrane to dry prior to staining.

  • Incubate in stain solution with agitation at room temperature for 4 min.

  • Remove stain solution and add 50 ml destain solution.  Incubate for 5 min with agitation at room temperature.

  • Rinse membrane in distilled water. Air dry.

 

F- Blocking- Signal to noise ratios can be improved by following the procedure listed below:

  • Remove membrane from gel surface and place in container.

  • Add 10 ml BIO-BLOCK per 100 cm2 of membrane surface.  Incubate at room temperature for 30 min. with agitation.

  • After blocking, TotalBlot PVDF membranes can be probed with antibodies and enzyme conjugates, followed by visualization with appropriate  colorimetric substrates.

 

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