Bio-Rad Sequi-Blo PVDF Membrane User Manual

Sequi-Blot PVDF

Membrane

for Pr otein Sequencing

Instruction

Manual

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

LIT240 Rev C

Table of Contents

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

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

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

Section 4 Electrophoretic Blotting

Section 5 Amino Acid Analysis by

Section 6 Protein Dectection .................16

Section 7 Technical Assistance .............18

Section 8 References ..............................19

Section 9 Product Information .............20

for Protein Sequencing ..........7

Hydrolysis of Membrane

Bound Proteins ......................13

Section 1
Introduction

The hydrophobicity of PVDF (polyvinyli­dene difluoride) membrane makes it an ideal
support for binding proteins in electrophoretic
and dot blotting applications. Proteins are tight­ly bound, and are quantitatively retained during
exposure to acidic, basic, or organic solvents.
Resistance to acidic and organic solvents which
would dissolve nitrocellulose or nylon mem­branes makes PVDF membrane an excellent
support for amino-terminal protein sequenc-

1,3

ing.

The unique manufacturing process used
for Bio-Rad’s Sequi-Blot PVDF membrane
insures higher protein binding capacity than
other commercially available PVDF products.
Higher capacity increases the likelihood of
sequencing proteins of interest, which makes
Sequi-Blot PVDF membrane the best choice for

1

1,2

4

sequence analysis of blotted proteins and pep­tides.

For use of PVDF membrane as an
immunoblotting support for western blot detec­tion, Bio-Rad offers Immun-Blot

®

PVDF mem­brane which features lower signal to
background results in this application than
Sequi-Blot membrane. However, it does not
exhibit the same high protein binding capacity,
so Sequi-Blot PVDF membrane is the best one
to use for protein sequencing.

Section 2
Membrane Wetting

Sequi-Blot PVDF membrane can be used
as a direct replacement for the membrane cur­rently being used in your sequencing 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 the PVDF membrane makes
it impossible to wet the membrane with aque­ous solutions. Methanol or an alternative organ­ic solvent is required to pre-wet the membrane
prior to equilibration in 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.

2

3

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 solu­tion. 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 aque-

ous 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. When the membrane is
dry, protein molecules will not bind tightly and
will be washed off in subsequent analysis steps.
For protein sequencing, the amount of protein
bound is critical. For this application it might be
better to try the direct adsorption method for
attachment of proteins.
sistently provides 90% binding of small
amounts of protein. In this method, up to
300 pmol of protein is suspended in 200 µl of
buffer and incubated with a small piece (5 x 9
mm) of wetted PVDF membrane for 1 hour

3

Direct adsorption con-

4

5

with agitation (rotary shaker) or 24 hours with­out. The incubation is carried out at 4 °C in a
siliconized glass tube. As the amount of protein
increases above 300 pmol, the percent of pro­tein bound will decrease.

Note: The high binding capacity of Sequi-Blot PVDF
membrane makes it diffi-cult to block in immunoassays.
Use 0.5% casein, non-fat dry milk, or BSA, or Immun­Blot PVDF membrane for protein immunoblotting appli­cations for best results.

Section 4
Electrophoretic Blotting
for Protein Sequencing

This protocol is based on the techniques
practiced by Dr. David Speicher in the Protein
Micro-chemistry Lab at The Wistar Institute.
Each of the precautions recommended below
reduces the potential of amino terminus block­ing during the gel purification and blotting
steps. Proteins and peptides larger than 10,000
daltons bind so strongly to PVDF that a
Polybrene coated glass fiber filter is not
required for optimal sequence analysis.
Elimination of the Polybrene coated filters
saves the time normally required for precycling,
and reduces Polybrene associated background
seen in the initial sequencing cycles.
Alternative protocols for electrophoresis and
blotting of proteins for sequence analysis,
including options for recovering peptides from

3

6

7

proteins with a blocked amino terminus, are
reviewed in bulletin 2212.

Follow your standard procedure for
Laemmli SDS-PAGE, observing the changes
outlined below (solution recipes are included
after the blotting section):

1. Use reagents and solvents of the highest

purity. Use of Bio-Rad’s electrophoresis

reagents without further purification is rec-

ommended.

2. Filter gel solutions, except running buffer,

with a 0.45 micron filter and store at 4 °C.

Store SDS stock solution at RT.

3. Solubilize samples with 2x or 5x solubiliz-

ing buffer with sucrose. Do not use urea in

the solubilizing buffer.

4. Heat samples with solubilizing buffer at

37 °C for 10-15 minutes prior to loading

onto the gel. Do not heat samples at 100 °C.

5. Allow the gel, including stacker, to poly­merize completely. Let the cast gel stand
for 24-72 hours at room temperature prior
to use.

6. Add 11.4 mg/l (0.1 mM) thioglycolate to
the upper running buffer prior to elec­trophoresis to scavenge reactive com­pounds left in the gel which cause
N-terminal blocking.

7. Include 5 µg of a sequence standard (i.e.
myoglobin or ß-lactoglobulin A) as a con­trol in one lane.

8

9

Transfer Protocol

These instructions are for use with a tank blot

®

apparatus such as the Trans-Blot

cell. Blotting
in a Trans-Blot cell is preferable to semi-dry
transfers for protein sequencing because tank
blotting is more quantitative with higher bind­ing yields. Follow the instructions provided
with your blotting cell for assembly.

1. For most proteins, use a Towbin buffer

with methanol (MeOH).

2. PVDF membrane should be clean and free

of wrinkles.

3. Wet the membrane following the protocol

in the membrane wetting section.

4. Make sure there are no bubbles between the

membrane and the gel.

5. After transfer, rinse the membrane three

times (5 minutes each) with distilled water.

6. Stain for 5 minutes with the PVDF CBB R-

250 membrane stain. Do not use the stan-

10

dard CBB gel stain. Destain for 10-15
minutes, or until background is light blue,
with PVDF destain solution. Avoid acetic

acid in the stain and destain, as it might
cause blockage of the amino terminus.

Solutions

Stock sample buffer solution (5x without urea):

5

0.5 M sucrose 42.78 g
15% SDS 37.5 g

312.5 mM Tris 9.5 g
10 mM Na

EDTA 0.925 g

2

Make to a volume of 225 ml with distilled
water. Heat gently to get into solution and
adjust to pH 6.9 with 1 N HCl. Adjust to a final
volume of 250 ml. Store at 4 °C in 10 ml
aliquots.

11

5x working sample buffer solution:

Add 100 µl of ß-mercaptoethanol and
100 µl of bromophenol blue (BPB) solution
(0.05% w/v) to 2 ml of 5x stock sample
buffer solution.

2x working sample buffer solution:

Add 1.5 ml of distilled water, 50 µl of
ß-mercaptoethanol, and 50 µl of BPB solu­tion (0.05% w/v) to 1 ml of 5x stock sample
buffer solution.

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
Prechill the buffer before use.

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.

2

Sequi-Blot PVDF membrane stain:

0.025% Coomassie

®

Blue R-250 dissolved

in a 40% MeOH solution

Sequi-Blot PVDF membrane destain:

50% MeOH solution

Section 5
Amino Acid Analysis by
Hydrolysis of Membrane

O.

2

Bound Proteins

Amino acid analysis requires homogeneous
proteins. SDS-PAGE electrophoresis provides
a convenient way to purify proteins. Blotting to
Sequi-Blot PVDF membrane provides a simple

12

13

way to isolate proteins separated in a gel.
Protein samples which have been immobilized
onto PVDF by either spotting, direct adsorp­tion, or electrophoretic blotting can be
hydrolyzed and subjected to amino acid analy-

6,7

sis.

Amino acid analysis of blotted proteins
using a post-column amino acid analyzer offers
a way to quantitate the sample present prior to
sequencing, as well as a way to determine the
A.A. composition of the proteins of interest.
This data can also be used to determine the effi­ciencies of the gel electrophoresis and blotting
systems being used for sequencing, whether the
protein of interest is present in enough quantity
to provide useful sequence data, or whether the
quantity of protein is not a problem and amino
terminal blockage might be the cause of
sequencing problems. For amino acid analysis:

1. Place the PVDF membrane piece contain-

ing the sample in a hydrolysis tube and add

200 µl of 6 N HCl containing 4% thiogly­colic acid.

2. Seal the tube in vacuo and hydrolyze for
16-24 hours at 110 °C.

3. Extract the liquid containing the amino
acids from the tube, and rinse the PVDF
membrane with an additional 50-100 µl of
6 N HCl.

4. Combine the rinse wash with the extraction
sample and evaporate to dryness.

5. Analyze samples with an amino acid ana­lyzer.

Note: As a blank control to account for background,
cut a piece of membrane the same size as the
band of interest from a corner of the membrane
and subject it to hydrolysis.

14

15

Section 6
Protein Detection

Staining

Proteins for sequencing can be detected
with Coomassie brilliant blue (CBB) R-250 or
with Bio-Rad’s Colloidal Gold Total Protein
Stain. CBB R-250 is the stain most commonly
used for detection of proteins prior to protein
sequence analysis. It is rapid (² 15–20 minutes),
sensitive, and does not interfere or react with
the reagents used in Edman degradation chem­istry. See page 13 for the CBB R-250 stain
preparation without acetic acid. If a protein can
not be detected by CBB staining, there will not
be enough present to sequence. See the Blotting
for Protein Sequencing section for a protocol
for CBB R-250 staining.

Section 7
For Technical Assistance

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). Technical representatives can
be contacted through your local Bio-Rad office.
For more information about the use of PVDF
membrane, request bulletin 2212.

16

17

Section 8
References

1. Matsudaira, P., J. Biol. Chem., 262, 10035-

10038 (1987).

2. Hildebrandt, E. and Fried, V., Anal. Biochem.,

177, 407-412 (1989)

3. Speicher, D. W., in Techniques in Protein
Chemistry (T. Hugli, ed.), Academic Press,
24–35 (1989).

4. Speicher, D. W., Mozdzanowski, J., Beam, K.
and Chem, D., J. Protein Chemistry, (1990).

5. Towbin, H., Staehelin, T. and Gordon, J., Proc.
Natl. Acad. Sci. USA, 76, 4350-4354 (1979).

6. Hulmes, J., Miedel, M. and Pan, Y., in
Techniques in Protein Chemistry (T. Hugli,
ed.), Academic Press, 7-16 (1989).

7. Nakagawa, S. and Fukuda, T., Anal. Biochem.,
181, 75-78 (1989).

Section 9
Product Information

Catalog
Number Product Description

Sequi-Blot PVDF Membrane

162-0180 Sequi-Blot PVDF Membrane,

10 x 15 cm, 10 sheets

162-0181 Sequi-Blot PVDF Membrane,

15 x 15 cm, 10 sheets

162-0182 Sequi-Blot PVDF Membrane,

20 x 20 cm, 10 sheets

162-0183 Sequi-Blot PVDF Membrane,

30 cm x 3 m, 1 roll

162-0185 Sequi-Blot PVDF Membrane,

20 x 20 cm, 3 sheets

162-0186 Sequi-Blot PVDF Membrane,

7 x 8.4 cm, 10 sheets

18

19

Catalog
Number Product Description

Blotting Equipment

170-3910 Trans-Blot Electrophoretic

Transfer Cell

170-3946 Trans-Blot Cell with Plate

Electrodes

170-3930 Mini Trans-Blot

®

Cell

Electrophoresis and Blotting Reagents

161-0400 Coomassie Brilliant Blue R-250,

10 g

170-6527 Colloidal Gold Total Protein

Stain, 500 ml

161-0305 Prestained SDS-PAGE

Standards, low range, 500 µl

161-0309 Prestained SDS-PAGE

Standards, high range, 500 µl

161-0318 Prestained SDS-PAGE

Standards, broad range, 500 µl

161-0326 Polypeptide SDS-PAGE

Standards, 200 µl

Catalog
Number Product Description

161-0304 SDS-PAGE Standards,

low range, 200 µl

161-0303 SDS-PAGE Standards,

high range, 200 µl

161-0317 SDS-PAGE Standards,

broad range, 200 µl

Coomassie is a trademark of ICI Organics.

20

21