Western blot transfer protocols

 

Transfer Conditions for Western blotting

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Since the introduction of the electrophoretic transfer protocol by Towbin et al. in 1979and re-named western blot by Burnette WN 19812,  there have been many modifications that assist better transfer of particular types of proteins. In addition, two main membrane types exist that further complicate the combinations of testing conditions. Fortunately, a lot of published work has created a database from which to pull optimized protocols. In this article, we have identified some useful protocols that apply to individual classes of proteins and have worked well in our labs. These are summarized into the following types (click to move to protocol):

1. Typical mid-range proteins.

2. Small electrophoretically mobile proteins such as prenylated small G-proteins.

3. Phosphorylated proteins.

4. High molecular weight proteins.

5. Acetylated proteins.

6. Sumoylated proteins.

7. Ubiquitinated proteins.

 

 

 

1. Typical mid-range proteins.

Useful notes

Over 70% of proteins fall into a typical category when considering western blot protocols. The standard conditions in the western blot protocol described below will be sufficient for efficient transfer and detection by quality antibodies. Two general preliminary guidelines are useful for setting the foundation for successful western blots these are 1) SDS must be removed from the separating gel prior to transfer of proteins that are less than 100 kDal, failing to do this will allow the protein to pass straight through the membrane because its charge has been modified to a higher pI than the pH of the buffer by the sulphate groups of SDS. This can be accomplished by the second recommendation, 2) a simple equilibrating step of the gel in transfer buffer for 10 min with agitation, prior to assembling the transfer setup. A great control sample would be a recombinant over-expressed protein from E.coli or higher eukaryote cell culture.

 

Required materials and buffers

  • Transfer buffer, One liter of 25 mM Tris Base (3.0 g/l), 195 mM glycine (14.4 g/l), and 15% (v/v) methanol, pH should be 8.30 after dissolving Tris base and glycine in 835 ml nanopure water. Make the solution fresh in a clean 1L bottle as the glycine is easily polymerized if left for a few weeks in a re-used bottle which leaves broad bands on the gel/blot.
  • PVDF membrane, 0.2 micrometer pore size, from EMD Millipore.
  • Gel sized Whatman 3M filter paper (two pieces).

 

Required equipment

Standard liquid based polyacrylamide gel transfer system from Biorad or GE Healthcare or other supplier (we have found most mini-gel blotting apparatus works well, but you should avoid rapid dry blotting equipment as these are difficult to optimize and have in general high backgrounds).

 

Protocol

a)   Prepare samples in 1x SDS-loading buffer and run on a 4-20% mini-gel with molecular weight markers and control sample. We recommend running 20 μg of TSA-treated cell lysate as a control.

b)   Carefully remove the gel from the pre-cast assembly and equilibrate the gel in transfer buffer with constant agitation 60-120 rpm for 15 min at room temperature.

c)    Prepare PVDF membrane by cutting to gel size and placing in 20 ml of 100% methanol.

d)   Assemble the blotting sandwich with coarse plastic filter supports provided by the electroblotter manufacturer. From negative to positive electrodes it will be coarse filter/support > filter paper > gel > PVDF membrane > filter paper > coarse filter/support, place in assembly and into electroblotting chamber.

Notes about the sandwich;

  • All components should be initially soaked in buffer, usually there is a tray provided by the electroblotter manufacturer for this purpose.
  • The PVDF membrane should be dipped once in transfer buffer for 2-3 seconds prior to placing on top of the gel.
  • Use a mini roller provided by the electroblotter company to roll across the sandwich after the second filter paper is added, this removes bubbles that would distort band transfer.

e)      Transfer the protein to a PVDF membrane for 60 min at 75 V, or 45 min for small proteins, or for proteins larger than 100 kDal refer to the large protein transfer protocol.

f)       After electro-transfer, wash the membrane for 1 min with TBS with constant 60-120 rpm agitation. The membrane is now ready to use for processing with block and antibody reagents (see western blot processing page for this technique or refer to the western blot processor manual(Cat.# WBM01) for an automated procedure.

g)      Alternatively for longer term storage up to 3 months, air dry the membrane at room temperature for 30 min. The membrane must be fully re-hydrated in TBST for 30 min. with agitation prior to the antibody processing procedure.  

h)    Processing membranes with blocking agents and antibodies can be achieved manually or by utilizing the GOBlot Western Blot processor (Cat. #WBM01) which ia an affordable alternative and can save you 3 hours per day.

 

2. Small electrophoretically mobile proteins.

Useful notes.

Small electrophoretically mobile proteins will rapidly move out of the gel and actually through a protein binding membrane without prior precautions to retain them. There are generally four approaches taken to reduce losses due to trans-migration through the gel, these are; 1) to reduce the methanol content which improves the hydrophobic nature of the surface of the protein, 2) reduce the time or transfer, 3) perform transfer in colder conditions, and 4) to dry the blot after electroblotting. These four modifications to the standard protocol are usually used altogether and allow 10-50 fold higher signal detection. 

Two other general points are that SDS must be removed from the separating gel prior to electroblotting, failing to do this will cause small proteins to pass straight through the membrane because its charge has been modified to a higher pI than the pH of the buffer by the sulphate groups of SDS. This can be accomplished by simply equilibrating the gel in transfer buffer for 10 min with agitation prior to assembling the transfer setup. A great control sample for an phosphoryl western blot is a cell culture treated with a broad spectrum phosphatase inhibitor such as 100 micromolar sodium vanadate for 4 h pre-lysis.

 

Required materials and buffers

  • Transfer buffer, One liter of 25 mM Tris Base (3.0 g/l), 195 mM glycine (14.4 g/l), and 15% (v/v) methanol, pH should be 8.30 after dissolving Tris base and glycine in 835 ml nanopure water. Make the solution fresh in a clean 1L bottle as the glycine is easily polymerized if left for a few weeks in a re-used bottle which leaves broad bands on the gel/blot.
  • PVDF membrane, 0.2 micrometer pore size, from EMD Millipore.
  • Gel sized Whatman 3M filter paper (two pieces).

 

Required equipment

Standard liquid based polyacrylamide gel transfer system from Biorad or GE Healthcare or other supplier (we have found all the mini-gel blotting apparatus works well, but you should avoid rapid dry blotting as these are difficult to optimize and have in general high backgrounds).

 

Protocol

a)    Prepare samples in 1x SDS-loading buffer and run on a 4-20% mini-gel with molecular weight markers and control sample. We recommend running 20 μg of Na-vanadate-treated cell lysate as a control.

b)   Carefully remove the gel from the pre-cast assembly and equilibrate the gel in transfer buffer with constant agitation 60-120 rpm for 15 min at room temperature.

c)    Prepare PVDF membrane by cutting to gel size and placing in 20 ml of 100% methanol.

d)   Assemble the blotting sandwich with coarse plastic filter supports provided by the electroblotter manufacturer. From negative to positive 
electrodes it will be coarse filter/support > filter paper > gel > PVDF membrane > filter paper > coarse filter/support, place in assembly and into electroblotting chamber.

Notes about the sandwich;

  • All components should be initially soaked in buffer, usually there is a tray provided by the electroblotter manufacturer for this purpose.
  • The PVDF membrane should be dipped once in transfer buffer for 2-3 seconds prior to placing on top of the gel.
  • Use a mini roller provided by the electroblotter company to roll across the sandwich after the second filter paper is added, this removes bubbles that would distort band transfer.

e)   Transfer the protein to a PVDF membrane for 60 min at 75 V, or 45 min for small proteins, or for proteins larger than 100 kDal refer to the large protein transfer protocol.

f)    After electro-transfer, wash the membrane for 1 min with TBS with constant 60-120 rpm agitation. The membrane is now ready to use for processing with block and antibody reagents (see western blot processing page for this technique or refer to the western blot processor manual(Cat.# WBM01) for an automated procedure.

g)   Alternatively for longer term storage up to 3 months, air dry the membrane at room temperature for 30 min. The membrane must be fully re-hydrated in TBST for 30 min. with agitation prior to the antibody processing procedure.  

h)    Processing membranes with blocking agents and antibodies can be achieved manually or by utilizing the GOBlot Western Blot processor (Cat. #WBM01) which ia an affordable alternative and can save you 3 hours per day.

 

 

3. Phosphorylated proteins.

Useful notes.

Phosphorylated proteins gain a negative charge at the expense of a hydroxyl group usually from modified serine, tyrosine or threonine amino-acids which may shift the overall charge (pI) of the molecule to the acidic end of the pH scale. This does not require modifying the transfer buffer because the protein’s pI will still be below the pH of the transfer buffer, pH 8.3. Another adaptation is to process samples rapidly on ice in the presence of phosphorylase and kinase inhibitors such that the level of phosphorylation is not affected by reverse hydrolysis of the phosphodiester bond which can be labile.  Two other general points are that SDS must be removed from the separating gel prior to transfer for proteins less than 100 kDal, failing to do this will allow the protein to pass straight through the membrane because its charge has been modified to a higher pI than the pH of the buffer by the sulphate groups of SDS. This can be accomplished by simply equilibrating the gel in transfer buffer for 10 min with agitation prior to assembling the transfer setup. A great control sample for an phosphoryl western blot is a cell culture treated with a broad spectrum phosphatase inhibitor such as 100 micromolar sodium vanadate for 4 h pre-lysis.

 

Required materials and buffers

  • Transfer buffer, One liter of 25 mM Tris Base (3.0 g/l), 195 mM glycine (14.4 g/l), and 15% (v/v) methanol, pH should be 8.30 after dissolving Tris base and glycine in 835 ml nanopure water. Make the solution fresh in a clean 1L bottle as the glycine is easily polymerized if left for a few weeks in a re-used bottle which leaves broad bands on the gel/blot.
  • PVDF membrane, 0.2 micrometer pore size, from EMD Millipore.
  • Gel sized Whatman 3M filter paper (two pieces).

 

Required equipment

Standard liquid based polyacrylamide gel transfer system from Biorad or GE Healthcare or other supplier (we have found all the mini-gel blotting apparatus works well, but you should avoid rapid dry blotting as these are difficult to optimize and have in general high backgrounds).

 

Protocol

a)    Prepare samples in 1x SDS-loading buffer and run on a 4-20% mini-gel with molecular weight markers and control sample. We recommend running 20 μg of Na-vanadate-treated cell lysate as a control.

b)   Carefully remove the gel from the pre-cast assembly and equilibrate the gel in transfer buffer with constant agitation 60-120 rpm for 15 min at room temperature.

c)    Prepare PVDF membrane by cutting to gel size and placing in 20 ml of 100% methanol.

d)   Assemble the blotting sandwich with coarse plastic filter supports provided by the electroblotter manufacturer. From negative to positive 
electrodes it will be coarse filter/support > filter paper > gel > PVDF membrane > filter paper > coarse filter/support, place in assembly and into electroblotting chamber.

Notes about the sandwich;

  • All components should be initially soaked in buffer, usually there is a tray provided by the electroblotter manufacturer for this purpose.
  • The PVDF membrane should be dipped once in transfer buffer for 2-3 seconds prior to placing on top of the gel.
  • Use a mini roller provided by the electroblotter company to roll across the sandwich after the second filter paper is added, this removes bubbles that would distort band transfer.

e)   Transfer the protein to a PVDF membrane for 60 min at 75 V, or 45 min for small proteins, or for proteins larger than 100 kDal refer to the large protein transfer protocol.

f)    After electro-transfer, wash the membrane for 1 min with TBS with constant 60-120 rpm agitation. The membrane is now ready to use for processing with block and antibody reagents (see western blot processing page for this technique or refer to the western blot processor manual(Cat.# WBM01) for an automated procedure.

g)   Alternatively for longer term storage up to 3 months, air dry the membrane at room temperature for 30 min. The membrane must be fully re-hydrated in TBST for 30 min. with agitation prior to the antibody processing procedure.  

h)    Processing membranes with blocking agents and antibodies can be achieved manually or by utilizing the GOBlot Western Blot processor (Cat. # WBM01) which ia an affordable alternative and can save you 3 hours per day.

 

 

4. High molecular weight proteins.

Useful notes.

High molecular weight (HMW) proteins have low mobility through polyacrylamide gels which reduces transfer by reducing the amount of protein that can reach the membrane under standard transfer conditions. There are several techniques that can be used to optimize the transfer by increasing mobility. These are three fold; 1) add SDS to the transfer buffer, 2) increase the time for transfer, these modifications should be tried before the third method which needs more titration for optimization. 3) introduce mild proteolysis to cleave the protein into smaller pieces.

The optimal amount of SDS is 0.025% (w/v) which is enough to coat a portion of the positive charged amino-acid side chains but not enough to completely interfere with binding to the membrane. The SDS must be in the equilibrating buffer prior to assembling the transfer sandwich, use 10 min with agitation prior to assembling the transfer setup. A great control sample is to use 100 nanograms of skeletal myosin as a well known large 240 kDal protein.

Protein hydrolysis can be of two varieties which are performed in the gel by washing in the appropriate solution/buffer, these are acid or protease treatment. An example acid treatment could be 10, 30, 100 or 300 min in 100 mM acetic acid, then equilibrating in transfer buffer prior to electroblotting. SDS is not required for this process. Protease treatment of the gel can be with 0,001, 0.01 and 0.1 units per ml of trypsin in pH 7.0 20 mM Tris-HCl, then equilibrating in transfer buffer prior to electroblotting. SDS is not required for this process.   

 

Required materials and buffers

  • Transfer buffer, One liter of 25 mM Tris Base (3.0 g/l), 195 mM glycine (14.4 g/l), 0.025% (w/v) SDS(0.25g/l) and 15% (v/v) methanol, pH should be 8.30 after dissolving Tris base and glycine in 835 ml nanopure water. Make the solution fresh in a clean 1L bottle as the glycine is easily polymerized if left for a few weeks in a re-used bottle which leaves broad bands on the gel/blot.
  • PVDF membrane, 0.2 micrometer pore size, from EMD Millipore.
  • Gel sized Whatman 3M filter paper (two pieces).

 

Required equipment

Standard liquid based polyacrylamide gel transfer system from Biorad or GE Healthcare or other supplier (we have found all the mini-gel blotting apparatus works well, but you should avoid rapid dry blotting as these are difficult to optimize and have in general high backgrounds).

 

Protocol

a)   Prepare samples in 1x SDS-loading buffer and run on a 4-20% mini-gel with molecular weight markers and control sample. We recommend running 20 μg of TSA-treated cell lysate as a control.

b)   Carefully remove the gel from the pre-cast assembly and equilibrate the gel in transfer buffer with constant agitation 60-120 rpm for 15 min at room temperature.

c)    Prepare PVDF membrane by cutting to gel size and placing in 20 ml of 100% methanol.

d)   Assemble the blotting sandwich with coarse plastic filter supports provided by the electroblotter manufacturer. From negative to positive 
electrodes it will be coarse filter/support > filter paper > gel > PVDF membrane > filter paper > coarse filter/support, place in assembly and into electroblotting chamber.

Notes about sandwich;

  • All components should be initially soaked in buffer, usually there is a tray provided by the electroblotter manufacturer for this purpose.
  • The PVDF membrane should be dipped once in transfer buffer for 2-3 seconds prior to placing on top of the gel.
  • Use a mini roller provided by the electroblotter company to roll across the sandwich after the second filter paper is added, this removes bubbles that would distort band transfer.

 

e)      Transfer the protein to a PVDF membrane for 120 min at 75 V.

f)       After electro-transfer, wash the membrane for 1 min with TBS with constant 60-120 rpm agitation. The membrane is now ready to use for processing with block and antibody reagents (see western blot processing page for this technique or refer to the western blot processor manual(Cat.# WBM01) for an automated procedure.

g)      Alternatively for longer term storage up to 3 months, air dry the membrane at room temperature for 30 min. The membrane must be fully re-hydrated in TBST for 30 min. with agitation prior to the antibody processing procedure.  

h)    Processing membranes with blocking agents and antibodies can be achieved manually or by utilizing the GOBlot Western Blot processor (Cat. #WBM01) which ia an affordable alternative and can save you 3 hours per day.

 

5. Acetylated proteins

Useful notes.

Acetylated proteins gain a negative charge at the expense of a positive charge usually from modified lysine or arginine amino-acids which may shift the overall charge (pI) of the molecule to the acidic end of the pH scale. This does not require modifying the transfer buffer because the protein’s pI will still be below the pH of the transfer buffer, pH 8.3. Another adaptation is to process samples rapidly on ice such that the level of acetylation is not affected by reverse hydrolysis of the acetyl group by deactylase enzymes or pH of the sample solution. Two other general points are that SDS must be removed from the separating gel prior to transfer for proteins less than 100 kDal, failing to do this will allow the protein to pass straight through the membrane because its charge has been modified to a higher pI than the pH of the buffer by the sulphate groups of SDS. This can be accomplished by simply equilibrating the gel in transfer buffer for 10 min with agitation prior to assembling the transfer setup. A great control sample for an acetyl western blot is a cell culture treated with a broad spectrum deacetylase inhibitor such as 1-5 micromolar trichlorostatin A (also called Trichostatin A) for 4 h pre-lysis.  

 

Required materials and buffers

  • Transfer buffer, One liter of 25 mM Tris Base (3.0 g/l), 195 mM glycine (14.4 g/l), and 15% (v/v) methanol, pH should be 8.30 after dissolving Tris base and glycine in 835 ml nanopure water. Make the solution fresh in a clean 1L bottle as the glycine is easily polymerized if left for a few weeks in a re-used bottle which leaves broad bands on the gel/blot.
  • PVDF membrane, 0.2 micrometer pore size, from EMD Millipore.
  • Gel sized Whatman 3M filter paper (two pieces).

 

Required equipment

Standard liquid based polyacrylamide gel transfer system from Biorad or GE Healthcare or other supplier (we have found all the mini-gel blotting apparatus works well, but you should avoid rapid dry blotting as these are difficult to optimize and have in general high backgrounds).

 

Protocol

a)   Prepare samples in 1x SDS-loading buffer and run on a 4-20% mini-gel with molecular weight markers and control sample. We recommend running 20 μg of TSA-treated cell lysate as a control.

b)   Carefully remove the gel from the pre-cast assembly and equilibrate the gel in transfer buffer with constant agitation 60-120 rpm for 15 min at room temperature.

c)    Prepare PVDF membrane by cutting to gel size and placing in 20 ml of 100% methanol.

d)   Assemble the blotting sandwich with coarse plastic filter supports provided by the electroblotter manufacturer. From negative to positive 
electrodes it will be coarse filter/support > filter paper > gel > PVDF membrane > filter paper > coarse filter/support, place in assembly and into electroblotting chamber.

Notes about the sandwich;

  • All components should be initially soaked in buffer, usually there is a tray provided by the electroblotter manufacturer for this purpose.
  • The PVDF membrane should be dipped once in transfer buffer for 2-3 seconds prior to placing on top of the gel.
  • Use a mini roller provided by the electroblotter company to roll across the sandwich after the second filter paper is added, this removes bubbles that would distort band transfer.

 

e)      Transfer the protein to a PVDF membrane for 60 min at 75 V, or 45 min for small proteins, or for proteins larger than 100 kDal refer to the large protein transfer protocol.

f)       After elecrto-transfer, wash the membrane for 1 min with TBS with constant 60-120 rpm agitation. The membrane is now ready to use for processing with block and antibody reagents (see western blot processing page for this technique or refer to the western blot processor manual(Cat.# WBM01) for an automated procedure.

g)      Alternatively for longer term storage up to 3 months, air dry the membrane at room temperature for 30 min. The membrane must be fully re-hydrated in TBST for 30 min. with agitation prior to the antibody processing procedure.  

h)    Processing membranes with blocking agents and antibodies can be achieved manually or by utilizing the GOBlot Western Blot processor (Cat. #WBM01) which ia an affordable alternative and can save you 3 hours per day.

 

6. Sumoylated proteins.

Useful notes.

Sumoylated proteins gain an 8 kDal protein by crosslinking to lysine or arginine amino-acid on the surface of the native protein which may shift the overall charge (pI) of the molecule to the acidic side of the pH scale. This does not require modifying the transfer buffer because the protein’s pI will still be below the pH of the transfer buffer, pH 8.3. Another adaptation is to process samples rapidly on ice such that the level of sumoylation is not affected by reverse hydrolysis of the linking bond by de-sumoylating enzymes or pH of the sample solution. Two other general points are that SDS must be removed from the separating gel prior to transfer for proteins less than 100 kDal, failing to do this will allow the protein to pass straight through the membrane because its charge has been modified to a higher pI than the pH of the buffer by the sulphate groups of SDS. This can be accomplished by simply equilibrating the gel in transfer buffer for 10 min with agitation prior to assembling the transfer setup. A great control sample for a sumoyl western blot is a cell culture treated with a broad spectrum desumoylation inhibitor such as 1-5 micromolar N-Ethylmaleimide (NEM) for 24 h pre-lysis.

 

Required materials and buffers

  • Transfer buffer, One liter of 25 mM Tris Base (3.0 g/l), 195 mM glycine (14.4 g/l), and 15% (v/v) methanol, pH should be 8.30 after dissolving Tris base and glycine in 835 ml nanopure water. Make the solution fresh in a clean 1L bottle as the glycine is easily polymerized if left for a few weeks in a re-used bottle which leaves broad bands on the gel/blot.
  • PVDF membrane, 0.2 micrometer pore size, from EMD Millipore.
  • Gel sized Whatman 3M filter paper (two pieces).

 

Required equipment

Standard liquid based polyacrylamide gel transfer system from Biorad or GE Healthcare or other supplier (we have found all the liquid based mini-gel blotting apparatus works well, but you should avoid rapid dry blotting as these are difficult to optimize and have generally high backgrounds).

 

Protocol

a)   Prepare samples in 1x SDS-loading buffer and run on a 4-20% mini-gel with molecular weight markers and control sample. We recommend running 20 μg of xxxxxxx-treated cell lysate as a control.

b)   Carefully remove the gel from the pre-cast assembly and equilibrate the gel in transfer buffer with constant agitation 60-120 rpm for 15 min at room temperature.

c)    Prepare PVDF membrane by cutting to gel size and placing in 20 ml of 100% methanol.

d)   Assemble the blotting sandwich with coarse plastic filter supports provided by the electroblotter manufacturer. From negative to positive 
electrodes it will be coarse filter/support > filter paper > gel > PVDF membrane > filter paper > coarse filter/support, place in assembly and into electroblotting chamber.

Notes about the sandwich;

  • All components should be initially soaked in buffer, usually there is a tray provided by the electroblotter manufacturer for this purpose.
  • The PVDF membrane should be dipped once in transfer buffer for 2-3 seconds prior to placing on top of the gel.
  • Use a mini roller provided by the electroblotter company to roll across the sandwich after the second filter paper is added, this removes bubbles that would distort band transfer.

 

e)   Transfer the protein to a PVDF membrane for 60 min at 75 V, or 45 min for small proteins, or for proteins larger than 100 kDal refer to the large protein transfer protocol.

f)    After electro-transfer, wash the membrane for 1 min with TBS with constant 60-120 rpm agitation. The membrane is now ready to use for processing with block and antibody reagents (see western blot processing page for this technique or refer to the western blot processor manual(Cat.# WBM01) for an automated procedure.

g)   Alternatively for longer term storage up to 3 months, air dry the membrane at room temperature for 30 min. The membrane must be fully re-hydrated in TBST for 30 min. with agitation prior to the antibody processing procedure.  

h)    Processing membranes with blocking agents and antibodies can be achieved manually or by utilizing the GOBlot Western Blot processor (Cat. #WBM01) which ia an affordable alternative and can save you 3 hours per day.

7. Ubiquitinated proteins.

Useful notes.

Ubiquitinated proteins gain an 8 kDal protein moiety at the expense of a positive charge usually from modified lysine or arginine amino-acids which may shift the overall charge (pI) of the molecule to the acidic side of the pH scale. This does not require modifying the transfer buffer because the protein’s pI will still be below the pH of the transfer buffer, pH 8.3. Another adaptation is to process samples rapidly on ice such that the level of ubiquitination is not affected by reverse hydrolysis of the linkage site by de-ubiquitinating enzymes or pH of the sample solution. Two other general points are that SDS must be removed from the separating gel prior to transfer for proteins less than 100 kDal, failing to do this will allow the protein to pass straight through the membrane because its charge has been modified to a higher pI than the pH of the buffer by the sulphate groups of SDS. This can be accomplished by simply equilibrating the gel in transfer buffer for 10 min with agitation prior to assembling the transfer setup. A great control sample for an acetyl western blot is a cell culture treated with a broad spectrum deacetylase inhibitor such as 1-5 micromolar N-Ethylmaleimide (NEM) for 18 h pre-lysis.

 

Required materials and buffers

  • Transfer buffer, One liter of 25 mM Tris Base (3.0 g/l), 195 mM glycine (14.4 g/l), and 15% (v/v) methanol, pH should be 8.30 after dissolving Tris base and glycine in 835 ml nanopure water. Make the solution fresh in a clean 1L bottle as the glycine is easily polymerized if left for a few weeks in a re-used bottle which leaves broad bands on the gel/blot.
  • PVDF membrane, 0.2 micrometer pore size, from EMD Millipore.
  • Gel sized Whatman 3M filter paper (two pieces).

 

Required equipment

Standard liquid based polyacrylamide gel transfer system from Biorad or GE Healthcare or other supplier (we have found all the mini-gel blotting apparatus works well, but you should avoid rapid dry blotting as these are difficult to optimize and have in general high backgrounds).

 

Protocol

a)   Prepare samples in 1x SDS-loading buffer and run on a 4-20% mini-gel with molecular weight markers and control sample. We recommend running 20 μg of TSA-treated cell lysate as a control.

b)   Carefully remove the gel from the pre-cast assembly and equilibrate the gel in transfer buffer with constant agitation 60-120 rpm for 15 min at room temperature.

c)    Prepare PVDF membrane by cutting to gel size and placing in 20 ml of 100% methanol.

d)   Assemble the blotting sandwich with coarse plastic filter supports provided by the electroblotter manufacturer. From negative to positive 
electrodes it will be coarse filter/support > filter paper > gel > PVDF membrane > filter paper > coarse filter/support, place in assembly and into electroblotting chamber.

Notes about the sandwich;

  • All components should be initially soaked in buffer, usually there is a tray provided by the electroblotter manufacturer for this purpose.
  • The PVDF membrane should be dipped once in transfer buffer for 2-3 seconds prior to placing on top of the gel.
  • Use a mini roller provided by the electroblotter company to roll across the sandwich after the second filter paper is added, this removes bubbles that would distort band transfer.

 

e)   Transfer the protein to a PVDF membrane for 60 min at 75 V, or 45 min for small proteins, or for proteins larger than 100 kDal refer to the large protein transfer protocol.

f)    After electro-transfer, wash the membrane for 1 min with TBS with constant 60-120 rpm agitation. The membrane is now ready to use for processing with block and antibody reagents (see western blot processing page for this technique or refer to the western blot processor manual(Cat.# WBM01) for an automated procedure.

g)   Alternatively for longer term storage up to 3 months at -20 C, air dry the membrane at room temperature for 30 min. The membrane must be fully re-hydrated in TBST for 30 min. with agitation prior to the antibody processing procedure.  

h)    Processing membranes with blocking agents and antibodies can be achieved manually or by utilizing the GOBlot Western Blot processor (Cat. #WBM01) which ia an affordable alternative and can save you 3 hours per day.

References:

1. Towbin H, Staehelin T, Gordon J. (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications"Proceedings of the National Academy of Sciences USA 76 (9): 4350–54. doi:10.1073/pnas.76.9.4350

2. Burnette WN. (1981). "'Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A". Analytical Biochemistry 112 (2): 195–203. doi:10.1016/0003-2697(81)90281-5