Bio-Rad Immun-Blot PVDF Membrane User Manual

Immun-Blot®PVDF

Membrane for

Protein Blotting

Instruction

Manual

Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547

4006127 Rev A

Table of Contents

Section 1 Introduction..............................1

Section 2 Membrane Wetting..................3

Section 3 Dot Blotting ..............................5

Section 4 Electrophoretic Blotting..........6

Section 5 Optimization of Blotting

Section 6 References ..............................10

Section 7 Product Information .............11

Conditions.................................8

Section 1
Introduction

PVDF (polyvinylidene difluoride) mem­brane was originally introduced to protein use as
an ideal medium for the harsh chemical environ­ment of N-terminal, or Edman degradation,
sequencing. The hydrophobicity of PVDF
makes it an ideal support for binding proteins in
electrophoretic and dot blotting applications.
Because of the hydrophobic nature of PVDF, it
does require a prewetting step in alcohol. The
high protein binding capacity, target retention,
and resistance to cracking has made PVDF an
appealing membrane for general lab techniques.

The two main applications for PVDF are N­Terminal Sequencing and Immunoblotting, both
of which benefit from the qualities of PVDF but
in different aspects of the membrane. While
sequencing work is concerned with retaining as

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much protein as possible, a western blot requires
good signal retention with very low background.
In order to provide the best possible membrane
for each technique, Bio-Rad offers two grades
of PVDF, Immun-Blot PVDF for western blot­ting, and Sequi-Blot PVDF for protein sequenc­ing.

can be added to the transfer buffer without
affecting the binding of the protein to PVDF.
The results are consistently clean, easy to read
blots. The physical strength of Immun-Blot
PVDF membrane means that it will not crack or
tear in common handling, and holds up under
repeated strip and reprobing applications.

Immun-Blot PVDF

The purpose of a blotting membrane is to
deliver good signal results while resisting back­ground and non-specific binding. Immun-Blot
PVDF Membrane is ideal for chemiluminescent
and colorimetric western blots because it retains
target protein very strongly, 140–150 µg pro-

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tein/cm
can obscure high sensitivity detection. Immun­Blot PVDF membrane retains proteins in any
transfer format; tank blotting, semi-dry blotting,
and dot blotting all deliver excellent results. For
proteins that are difficult to transfer, 0.1% SDS

membrane, but resists background that

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Section 2
Membrane Wetting

Immun-Blot PVDF membrane can be used
as a direct replacement for the membrane cur­rently being used in your blotting protocol. No
changes are required in the procedure, but the
special steps given below are required to pre­pare the membrane for blotting. The hydropho­bicity of PVDF makes it impossible to wet the
membrane with aqueous solutions. Methanol or
an alternative organic solvent is required to pre­wet the membrane prior to equilibration in

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transfer buffer. After equilibration, the mem­brane can be used in a semi-dry, tank, or capil­lary blotting system with any acidic or basic
blotting buffer.

Note: Always handle membranes using gloves or
forceps to prevent contamination.

1. Immerse the membrane in 100% methanol
for a few seconds, until the entire mem­brane is translucent. In methanol, it wets
immediately. (Solutions containing 50%
methanol concentration can be used to pre­wet the membrane.)

2. Transfer the wetted membrane to a vessel
containing transfer buffer or water.
Incubate in buffer until it is equilibrated
(² 2–3 minutes). The membrane will float
on the surface of the buffer until complete­ly equilibrated. After it is equilibrated it can
be easily submerged into the aqueous solu-

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tion. At this point, the membrane is ready
to bind proteins in any blotting application.

3. After the membrane has been wetted with
buffer, do not allow it to dry (white spots
will form where the membrane is dry).
Protein will not bind to the dried mem­brane, and dry spots will not rewet in aque­ous solutions. If the membrane becomes
dry prior to blotting, repeat steps 1 and 2 to
rewet it.

Section 3
Dot Blotting

Dot blotting requires special precautions to

insure that protein is bound to the PVDF mem­brane before it dries. Directly spot protein to the
wetted membrane and allow it to dry. Vacuum
assisted dot or slot blotting is not recommend­ed, due to the potential for the PVDF to dry out
before the proteins are bound.

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Section 4
Electrophoretic Blotting

Immun-Blot PVDF membrane can be used
with a variety of transfer equipment, including
Bio-Rad’s Trans-Blot cell, Mini Trans-Blot
cell, and Trans-Blot SD Semi Dry cell. In gen­eral, tank blotting is more quantitative with
higher binding yields than semi dry transfers.
However, very good results can be obtained
when a semi dry apparatus is used with a well
optimized gel and transfer system. The semi
dry transfer is much faster, with transfer times
as low as 15 minutes versus tank blotting
which requires 1 to 3 hours for transfers.

1. For most proteins, use a Towbin buffer

with methanol (MeOH).

2. For proteins that resist transfer out of the

gel, up to 0.1% SDS can be added to the

transfer buffer. This stabilizes the effect of

methanol in stripping SDS off the proteins.

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SDS imparts a net negative charge to the
proteins and helps facilitate movement out
of the gel. Binding of proteins to Immun­Blot PVDF membrane is not affected by the
addition of this amount of SDS.

3. PVDF should be clean and free of wrinkles.

4. Wet the membrane following the protocol
in the membrane wetting section.

5. Make sure there are no bubbles between the
membrane and the gel.

6. After transfer, rinse the membrane three
times (5 minutes each) with distilled water.

Solutions

1

Towbin buffer:

25 mM Tris 3.03 g
192 M glycine 14.4 g
20% methanol 200 ml

Adjust volume to 1 liter with dd H

O.

2

Prechill the buffer before use.

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Towbin/SDS buffer:

Towbin buffer as above with addition of up
to 0.1% SDS

Note: Do not add acid or base to adjust pH. The
buffer will range from pH 8.1 to 8.5, depending on the
quality of Tris, glycine, dd H
Methanol should be analytical reagent grade, as metallic
contaminants in low grade methanol will plate on the
electrodes.

O, and methanol.

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Section 5
Optimization of Blotting
Conditions

Immun-Blot PVDF membrane is a very
versatile blotting membrane. It can be used with
any of the blotting procedures that are used for
immunospecific antigen detection, or total pro­tein staining. Use the protocol developed for
your lab. If you do not have a standard protocol,
most blotting reagent kits will have a good pro-

tocol. Bio-Rad’s Immun-Blot colorimetric blot­ting kits, or Immun-Star

™

chemiluminescent

kits have excellent blotting protocols.

To minimize non-specific background, the
blocking step and antibody titers are very
important optimization steps. For blocking, we
recommend casein or non-fat dry milk. Typical
concentrations are from 0.2% to 5%. Antibody
dilution factors vary by the manufacture of the
antibody, the specificity, and the purity. To
optimize blot conditions, cut the PVDF into
strips, wet the membrane, and spot the test pro­tein directly to the membrane. Use different
blocking concentrations and antibody titers to
test the outcomes of these different conditions.
Once the blocking and antibody conditions are
set, you should have good signal to noise results
on the real electrophoretic blots.

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Note: The membrane can be stored after proteins have
been transferred, but prior to immunoblotting. Store it
dry and rewet it when needed. To dry the membrane,
place it on filter paper and let it dry at room temperature
for a few hours. When you are ready to analyze the blot­ted samples, rewet the membrane by placing it in 100%
methanol or into a staining solution that contains at least
50% MeOH.

In the U.S., technical service is available by
calling 1-800-4BIORAD (1-800-424-6723).
Our Technical Service representatives are
available to answer your questions from 8 AM
to 5 PM (PST).

Section 6
References

1. Towbin, H., Staehelin, T. and Gordon, J., Proc.

Natl. Acad. Sci. USA, 76, 4350-4354 (1979).

Section 7
Product Information

Catalog
Number Product Description

Immun-Blot PVDF Membrane

162-0174 7 x 8.4 cm, 10 sheets
162-0175 10 x 15 cm, 10 sheets
162-0176 20 x 20 cm, 10 sheets
162-0177 26 cm x 3.3 m, 1 roll

Blotting Equipment

170-3910 Trans-Blot Electrophoretic

170-3946 Trans-Blot Cell with Plate

170-3930 Mini Trans-Blot
170-3940 Trans-Blot SD Semi-Dry

Transfer Cell

Electrodes

Transfer Cell

®

Cell

10

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Catalog
Number Product Description

Blotting Reagents

161-0305 Prestained SDS-PAGE

161-0309 Prestained SDS-PAGE

161-0318 Prestained SDS-PAGE

161-0324 Kaleidoscope Prestained

170-6527 Colloidal Gold Total Protein

Related Blotting Detection Products

Standards, low range, 500 µl

Standards, high range, 500 µl

Standards, broad range, 500 µl

Standards, 500 µl

Stain, 500 ml

Immun-Blot colorimetric detection kits
Premixed Substrate Reagents for Colorimetric
Detection
Immun-Star chemiluminescent detection kits

In the US, call Bio-Rad at 1-800-4BIO­RAD (1-800-424-6723), or contact your local
office for more information on Bio-Rad blot­ting equipment and reagents.

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