Blotting Proteins fromPolycrylamide Gels

Section XI: Blotting Proteins from Polyacrylamide Gels

In This Section
Introduction
Semi-dry Blotting PAGEr® Gold Precast Gels
Tank Blotting PAGEr® Gold Precast Gels
References

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Section XI: Blotting Proteins from Polyacrylamide Gels

Introduction
Protein transfer efficiency in blotting applications isdependent upon multiple factors, including gel percentage,
gel thickness, protein size, transfer conditions (e.g.,buffer and voltage), and type and quality of membrane.

Blotting Proteins fromPolycrylamide Gels

Choosing the appropriate membrane
Nitrocellulose
Hydrophobic binding
General purpose membrane
Low backgroundLow strength
Becomes brittle if baked

To achieve optimal transfer efficiency, transfer conditions
must be adjusted to address these varying factors.

PVDF
Hydrophobic binding

Nylon
Hydrophobic & electrostatic binding

SDS tolerantHigh background
High strength
Suitable for protein sequencing

Stable if baked
High background
High strength
Least suitable for Western transfer

Transfer solutions
Formula for Towbin transfer solution:
Towbin Transfer Solution
1X Working Solution 		
25 mM Tris base 			
192 mM Glycine			
0.1% SDS											20% Methanol							Amount for 1X Working Solution
30.3 g Tris base144.1 g Glycine
10.0 gAdjust volume to 8 liters with distilled water
Measure, but do not adjust pH; it should be approximately 8.2 to 8.4
2 L Methanol
Adjust volume to 10 liters with distilled water

It may be necessary to lower the concentrations ofmethanol, SDS or both to obtain the optimal balance oftransfer and binding efficiency. The table below outlines
the effects that SDS and methanol have on proteintransfer.
SDSImproves transfer of proteins >60 kDa

MethanolImproves binding efficiency

Decreases binding efficiency
Not compatible with nylon membranes
Include 0.1% - 0.2% in transfer buffer

Decreases transfer efficiency
Do not soak gel in transfer buffer prior to blotting
Include 20% in transfer buffer

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Section XI: Blotting Proteins from Polyacrylamide Gels

Tips for increasing transfer efficiency—— Use the lowest concentration gel that will resolve theprotein(s) of interest.
—— Avoid using gels ≥1 mm thick. Thick gels may require
longer blotting times.
—— Decrease the concentration of methanol to optimize
transfer efficiency of proteins >150 kDa.
—— Small proteins tend to transfer more easily than large
proteins. Longer transfer times may be used to ensurecomplete transfer of large proteins (>60 kDa), proteinsfrom native gels, and thicker gels.
—— Use two membranes if transferring for an extended
period of time (>1 hour). The second membrane willbind any protein that may transfer through the first.
This can be verified by membrane staining.

—— Use ProSieve® Color Protein Markers (see page 51)
to confirm that transfer has occurred to the membraneand not the filter paper.
—— Use a chopping motion when removing the well andfoot area. Slicing the gel may cause tearing.
—— Gently roll out air bubbles between transfer stacklayers using a wet glass rod or pipette.

Blotting Proteins from
Polycrylamide Gels

Returnto Main—— If the gel sticks to the filter paper or membrane after
transfer, soak for 5 - 15 minutes in water then gently
peel the filter paper away.
—— If proteins are left in the gel after following
recommended transfer conditions, increasing thevoltage by no more than 5 volts may be helpful.

Section XI: Blotting Proteins from Polyacrylamide Gels
Semi-dry Blotting PAGEr® Gold Precast Gels
This protocol is for use with the BioRad® Trans-Blot®
Semi-Dry Cell. If using another manufacturer’s blotting
apparatus, follow manufacturer’s instructions for use.
Tips for Semi-Dry Blotting

Note: Transfer membranes should be handled at the edges
with gloves worn.
5.

Soak membrane, blotting paper and filter paper for
5 - 10 minutes in 1X transfer solution.

6.

To make a semi-dry blotting sandwich, stack in thefollowing order:—— To prevent the stack from drying out, add extra transfersolution (2 ml - 5 ml) to the top layer of filter paper
before closing the lid on the blotting apparatus.

6a.

Mask

6b.

Prewet extra thick blot paper

6c.

Prewet nitrocellulose membrane

6d.

Polyacrylamide gel.

6e.

Prewet Whatman® Grade 114 or 54 Filter Paper

Procedure for semi-dry blotting

6f.

Prewet extra thick blot paper

Note: Nitrocellulose or PVDF membranes arerecommended for this method and should be wetted inmethanol prior to wetting in transfer solution.

6g.

Top platen (stainless steel cathode), and safety
cover—— Center the gel on the membrane. Occasionally, thegel will overlap the membrane and stick to the filter
paper below. If this occurs, gently break the seal witha scalpel.

7.

Turn on the power and transfer at 25 volts (constant) at

400 mA for:1.

Electrophorese gel following standard procedure.

2.

Carefully trim off stacking gel and bottom foot from thegel.		

60 minutes for 10 kDa - 100 kDa proteins		

90 minutes for 100 kDa - 300 kDa proteins

3.

Soak gel for 20 minutes in chilled 1X transfer solution.

4.

Cut the filter paper, blot paper and membrane to the size ofthe gel.

 ote: Optimized conditions will be required for different
Nproteins or different membranes.
Continued on next page.

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Section XI: Blotting Proteins from Polyacrylamide Gels
Semi-dry Blotting PAGEr® Gold Precast Gels — continued–– Transfer membrane■■ Materials and Reagents

Blotting Proteins fromPolyacrylamide Gels–– Mask–– Scalpel or razor blade–– A piece of GelBond® Film (see page 61), or similar
polyester film, the size of the bottom anode, witha rectangular hole the size of the gel cut out of thecenter. The purpose of the mask is to focus current
through the gel stack.

–– Scissors–– Glass rod or pipette–– 1XTowbin transfer solution at 4°C–– Shallow tray for soaking membranes and filter paper–– Whatman® Grade 114 or 54 Filter Paper–– Extra thick blot paper

Section XI: Blotting Proteins from Polyacrylamide Gels
Tank Blotting PAGEr® Gold Precast Gels

For hydrophobic proteins or proteins >100 kDa, tankblotting is preferable to semi-dry blotting because
prolonged transfers are possible without gel drying. For
more detailed information and protocols concerning tankblotting, consult the blotting apparatus manufacturer’s
instructions.

6a.

Cathode unit

6b.

1 prewet fiber pad

6c.

1 sheet prewet extra thick blot paper

6d.

1 sheet prewet Whatman® 114 or 54 Blotting
Paper

Procedure for tank blotting

6e.

Polyacrylamide gel

Note: Transfer is performed at 4°C.
Note: Nitrocellulose or PVDF membranes arerecommended for this method and should be wettedin methanol prior to wetting in transfer solution.

6f.

Prewet nitrocellulose membrane

6g.

1 sheet prewet extra thick blot paper

6h.

1 prewet fiber pad

6i.

Anode unit

1.

Electrophorese gel following standard procedures.

2.

Carefully trim off stacking gel and bottom foot from thegel.

3.

Soak gel for 20 minutes in chilled 1X Transfer solution.

Cut the filter paper, blotting paper and membrane to thesize of the gel.
Note: Transfer membranes should be handled at theedges with gloves worn.
4.

5.

S oak fiber pads, nitrocellulose membrane and blotting
paper for 2 minutes in 1X Transfer solution.

6.

To make a tank blotting sandwich, stack in the following
order:

7.

Place in tank with nitrocellulose membrane closest toanode (+).

8.

Cover with chilled 1X Transfer solution.

9.

Turn on the power and transfer by running at 100 volts(constant) at 400 mA for:90 minutes for 10 kDa–100 kDa proteins
120 minutes for 100 kDa–300 kDa proteins

Note: Optimized conditions will be required for different
proteins or different membranes.

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www.lonza.com/researchSection XI: Blotting Proteins from Polyacrylamide Gels
Tank Blotting PAGEr® Gold Precast Gels - continued■■ Materials–– Whatman® Grade 114 or 54 Filter Paper–– Extra thick blot paper–– Transfer membrane–– Fiber or foam pad–– Scissors–– Glass rod or pipette–– Scalpel or razor blade–– 1X Towbin transfer solution at 4°C–– Shallow tray for soaking membranes and filter paper
Monitoring protein transferProtein transfer can be monitored by staining the gelfollowing transfer (see page 166), and/or staining themembrane. Membrane staining should only be performedwhen duplicate samples have been run on a gel and themembrane can be cut in half, or when a second membrane
has been used. Stains commonly used for this purpose
include SYPRO® Ruby Protein Blot Stain, (see page 55),
India ink stain or colloidal gold stain. Described below is
the use of the GE Healthcare AuroDye® Forte Kit which is acolloidal gold stain.

Staining membranes with the GE Healthcare
AuroDye® Forte Kit
This method is compatible with PVDF and nitrocellulose
membranes. For more detailed information and protocols,
consult the instructions provided with the kit.
1.

Place the membrane in a clean glass dish.

2.

Add 1X PBS, 0.3% Tween 20.

3.

Shake slowly on a shaker for 30 minutes at 37°C.

4.

Remove solution and replace with fresh 1X PBS, 0.3%Tween® 20.

5.

Shake slowly on a shaker for 5 minutes at room
temperature.

6.

Repeat steps 4 –5 two more times.

7.

Briefly rinse the membrane in distilled water.

8.

Place the membrane in a thermal seal pouch with 15 ml 20 ml AuroDye® Forte Kit for 2 - 4 hours at room
temperature.

9.

After fully developed, rinse briefly with distilled waterand air dry.

Blotting Proteins fromPolyacrylamide Gels

Returnto Main■■ Materials and Reagents–– Previously blotted membrane–– Orbital shaker set to 37°C–– Orbital or rocking platform shaker–– Glass dish–– Thermal seal pouch and sealing unit–– Distilled water–– 1X PBS, 0.3% Tween® 20References
Ausubel, F.M., et al., Current Protocols in Molecular Biology, Wiley & Sons, 1995.
Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Laboratory Press, 1989.

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Notes

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