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Protein Transfer

After separation of proteins by PAGE, the next step is to transfer the proteins from the gel to a solid support membrane, usually made of a chemically inert substrate such as nitrocellulose or polyvinylidene difluoride (PVDF).

Blotting Instruments Transfer Membranes Confirmation of Protein Transfer

Electrotransfer is the most commonly used method of transfer. Two types of electrotransfer, wet transfer and semidry transfer, are commonly used. GE Healthcare provides tank blotting instruments for high performance wet transfer as well as semidry blotting instruments.

During wet transfer, the gel and membrane are fully immersed in transfer buffer and current is applied in the direction of gel to membrane. Wet transfer is recommended for larger proteins or when it is important to obtain the best possible transfer efficiency. Our blotting instruments using wet transfer include: miniVE, TE22, and TE62.

For semidry transfer, several layers of filter paper soaked in transfer buffer are used to sandwich the gel and membrane. The sandwich is placed between two glass plates that form an anode and cathode when electric current is applied. Semidry transfer is faster than wet transfer and consumes less buffer. Our blotting instruments using semidry transfer include: TE70, TE77, and Multiphor II with NovaBlot Kit.

Electrically conducting transfer buffer provides electrical continuity between the electrodes of the transfer apparatus. In addition, the buffer provides a chemical environment that maintains protein solubility without interfering with its capacity to bind the membrane.

Towbin buffer is the most commonly used buffer in wet transfer, and consists of Tris and glycine. The pH of this buffer is 8.3, which is higher than the isoelectric point (pI) of most proteins, and ensures migration of negatively charged proteins (coated with SDS) toward the anode.

The degree to which molecules bind to a membrane is greatly influenced by the chemistry of the membrane itself as well as pore size, which ranges from 0.05 to 10 µm in diameter. A membrane with many small pores has a larger binding surface than one with larger pores, and thus generally has a higher binding capacity.

It should be noted that although protein conformation and buffer composition also affect binding capacity, the overall sensitivity of the assay depends on the amount of immobilized protein that is presented to the primary antibody. Nitrocellulose and PVDF membranes are the most common types of membranes used for Western blotting, although nylon is also sometimes used.

Table 1. Some important characteristics of membrane used in Western blotting

Membrane Interaction mode Optimal immobilization conditions Staining options Advantages Disadvantages
Nitrocellulose Non-covalent or hydrophobic High salt/low methanol Amino black Highly versatile Fragile unless supported with polyester support web  
      Aniline blue black Low background  
      Ponceau S    
      Colloidal gold    
      Fast green    
PVDF Dipole and hydrophobic interaction Prewet in methanol before using in aqueous buffers Amino black High protein binding capacity  
      Silver Mechanical strength  
      India Ink Chemical stability  
      Coomassie Brilliant Blue    
      Colloidal gold    
      Ponceau S    

 

Table 2. Suitability of membrane materials for use with different detection reagents

Nitrocellulose membranes PVDF membranes
  Amersham
Protran Premium
Amersham Protran Amersham Protran Supported Amersham
Hybond LFP1
Amersham Hybond Amersham
Hybond SEQ
Amersham ECL +++ ++ + +++ ++
Amersham ECL Prime ++/+++ ++ Nt +++ ++
Amersham ECL Plex +++ ++ +++ - -
ECF + - Nt +++ -
Colorimetric +++ ++ Nt ++ ++
Radioactive + ++ Nt ++ ++

+ = Suitable, ++ = Recommended, +++ = Highly recommended, - = Not recommended, nt = Not tested. ¹Amersham Hybond LFP PVDF is developed for fluorescence and has not been validated for other detection reagents.

Several methods allow you to test whether all proteins have transferred from the gel to the membrane. Total protein stains can be used to stain either the gel or membrane electrotransfer.

The use of visible, prestained molecular weight markers such as Rainbow Markers or DualVue Markers is a simple and convenient way to gauge the transfer of all proteins across the range of molecular weights of proteins in the gel, as high and low molecular weight proteins migrate with different efficiencies under similar electrophoretic conditions.

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