Western BLoT Stripping Buffer 使用说明书

Cat. #

T7135A

For Research Use

Western BLoT Stripping Buffer

Product Manual

v201510Da

Western BLoT Stripping Buffer

Cat. #T7135A

v201510Da

Table of Contents

I. Description ..........................................................................................................3

II. Components .......................................................................................................3

III. Storage ..................................................................................................................3

IV. Materials Required but not Provided ........................................................3

V. Precautions ..........................................................................................................3

VI. Protocol .................................................................................................................4

VII. Experimental Example ....................................................................................5

VIII. Related Products ...............................................................................................6

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Western BLoT Stripping Buffer

I. Description

Cat. #T7135A

v201510Da

The Western BLoT Stripping Buffer is a reagent that can remove primary and secondary antibodies

from Western blot membranes. After treatment with the Stripping Buffer, the membrane can be re-

used; it is possible to probe the membrane with either a different concentration of primary antibody

or with an entirely different primary antibody.

With this product, the antibody removal reaction proceeds under relatively mild conditions (room

temperature, 30 minutes), and therefore there is very little loss of immobilized protein from the

membrane. When using a PVDF membrane, the same membrane can be stripped and re-probed

2 - 5 times.

II. Components

Western BLoT Stripping Buffer 500 ml

Room temperature

III. Storage

IV. Materials Required but not Provided

Western blot membrane developed using chemiluminescence

・Western BLoT HRP Substrate series (Cat. #T7101A-T7104A) can be used for detection

Wash buffer (TBS or PBS containing 0.05% Tween 20)

・Tris Buffered Saline with Tween 20 (TBS-T) Tablets, pH7.6 (Cat. #T9142)

・Phosphate Buffered Saline with Tween 20 (PBS-T) Tablets, pH7.4 (Cat. #T9183)

Platform shaker

Reagents for Western blot detection (primary antibody, labeled secondary antibody, chemi-

luminescent detection reagent)

CCD camera or equipment and reagents for autoradiography.

V. Precautions

The following are precautions for using this product. Read before use.

1. Use this product for membranes detected with a chemiluminescence. This product can

not be used for membranes detected using chromogenic substrates such as TMB or DAB.

2. If the affinity of the antibody for the antigen is very strong, the antibody may not be

completely stripped from the membrane.

3. Once the membrane has been used for detection, do not allow the membrane to dry.

Store membranes submerged in PBS-T or TBS-T at 4℃.

4. A white precipitate may form if the buffer is stored at a low temperature. In this case,

warm in a water bath at 37℃ and shake gently to completely dissolve the precipitate

before use.

5. This product is acidic. When using, wear a lab coat, safety goggles, and gloves.

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Western BLoT Stripping Buffer

VI. Protocol

Cat. #T7135A

v201510Da

1. After chemiluminescent detection, wash the membrane in PBS-T or TBS-T buffer for

5 minutes. Discard the wash buffer.

2. Add Western BLoT Stripping Buffer so that the membrane is completely submerged.

Shake gently for 15 - 30 minutes at room temperature. Use approximately 0.25 ml of the

Stripping Buffer solution per cm

2

of membrane, and ensure that the membrane is com-

pletely immersed in the buffer during shaking.

3. Discard the Stripping Buffer. Wash the membrane for 5 minutes with PBS-T or TBS-T buf-

fer.

4. Block the membrane and perform immunodetection using a new antibody.

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Western BLoT Stripping Buffer

VII. Experimental Example

Cat. #T7135A

v201510Da

PVDF membranes that were detected with Western BloT Chemiluminescence HRP Substrate

(Cat. #T7101A) were stripped using the Western BLoT Stripping Buffer and re-probed. A

reaction using TBS-T instead of the stripping buffer was included as a negative control.

1．β-actin detection (first blot)

1 2 3 4 51 2 3 4 5

Lane

1：

2：

3：

4：

5：

HeLa Cell Lysate 10.0 μg

HeLa Cell Lysate 5.0 μg

HeLa Cell Lysate 2.5 μg

HeLa Cell Lysate 1.25 μg

HeLa Cell Lysate 0.625 μg

PVDF membrane

Nonfat milk/TBS-T, room

temperature, 60 min.

TBS-T

anti-β- actin (mouse IgG)

anti-mouse IgG-HRP

β-actin

Stripping (0.25 ml/cm

2

membrane, room

temperature, 30 min.)

Membrane:

Blocking:

Wash buffer:

Primary Ab:

Secondary Ab:

2．GAPDH detection (reprobe #1)

1 2 3 4 51 2 3 4 5

←

β-actin

GAPDH

Control (TBS-T)Stripping Buffer

Stripping (0.25 ml/cm

2

membrane, room temperature, 30 min.)

Wash buffer:

Primary Ab:

Secondary Ab:

TBS-T

anti-GAPDH (mouse IgG)

anti-mouse IgG-HRP

3．α-Tubulin detection (reprobe #2)

1 2 3 4 51 2 3 4 5

Wash buffer:

Primary Ab:

Secondary Ab:

TBS-T

anti-α-Tubulin (mouse IgG)

anti-mouse IgG-HRP

←

β-actin

←

GAPDH

α-Tubulin

Control (TBS-T)

Stripping Buffer

Stripping (0.25 ml/cm

2

membrane, room temperature, 30 min.)

4．Detection of residual antibody

1 2 3 4 5

1 2 3 4 5

←

α-Tubulin

←

β-actin

←

GAPDH

Control (TBS-T)

Stripping Buffer

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Wash buffer:

Secondary Ab:

TBS-T

anti-mouse IgG-HRP

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Western BLoT Stripping Buffer

VIII. Related Products

Cat. #T7135A

v201510Da

Western BLoT Chemiluminescence HRP Substrate (Cat. #T7101A/B)

Western BLoT Quant HRP Substrate (Cat. #T7102A/B)

Western BLoT Hyper HRP Substrate (Cat. #T7103A/B)

Western BLoT Ultra Sensitive HRP Substrate (Cat. #T7104A/B)

Western BLoT Immuno Booster (Cat. #T7111A)

Western BLoT Immuno Booster PF (Cat. #T7115A)

Western BLoT Rapid Detect (Cat. #T7121A)

Tris-Glycine-SDS Buffer (TG-SDS) Powder, pH8.3 (Cat. #T9101)

Tris-Glycine Buffer (TG) Powder, pH8.3 (Cat. #T9102)

CLEARLY Protein Ladder (Unstained) (Cat. #3453A/B)

CLEARLY Stained Protein Ladder (Cat. #3454A/B)

Phosphate Buffered Saline with Tween20 (PBS-T) Tablets, pH7.4 (Cat. #T9183)

Tris Buffered Saline with Tween20 (TBS-T) Tablets, pH7.6 (Cat. #T9142)

Western BLoT Blocking Buffer (Fish Gelatin) (Cat. #T7131A)

Western BLoT Blocking Buffer (Protein Free) (Cat. #T7132A)

NOTE : This product is for research use only. It is not intended for use in therapeutic or diagnostic

procedures for humans or animals. Also, do not use this product as food, cosmetic, or

household item, etc.

Takara products may not be resold or transferred, modified for resale or transfer, or used

to manufacture commercial products without written approval from TAKARA BIO INC.

If you require licenses for other use, please contact us by phone at +81 77 565 6973 or

from our website at .

Your use of this product is also subject to compliance with any applicable licensing

requirements described on the product web page. It is your responsibility to review,

understand and adhere to any restrictions imposed by such statements.

All trademarks are the property of their respective owners. Certain trademarks may not be

registered in all jurisdictions.

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