西姆伯意大利完整用户手册(T7101Q产品)说明书

Cat. #

T7101Q

For Research Use

Western BLoT

Chemiluminescence

HRP Substrate Sample

Product Manual

v201211

Western BLoT Chemiluminescence

HRP Substrate Sample

Cat. #T7101Q

v201211

Table of Contents

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

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

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

IV. Materials Required but Not Provided .......................................................4

V. Precautions ..........................................................................................................5

VI. Protocol .................................................................................................................6

VII. Troubleshooting ................................................................................................7

XIII. Related Products ...............................................................................................8

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Western BLoT Chemiluminescence

HRP Substrate Sample

I. Description

Cat. #T7101Q

v201211

Western BLoT Chemiluminescence HRP substrate is a horseradish peroxidase (HRP) substrate

specially developed for X-ray film imaging of Western blots. This product is part of the Western BLoT

HRP substrate series and produces a strong signal for high sensitivity with short exposure times. The

signal from Western BLoT Chemiluminescence HRP substrate is long-lasting, allowing the blot to be

re-imaged.

For detection using a CCD imager or for applications requiring higher sensitivity, we recommend

using other products in the Western BLoT HRP Substrate series:

Western BLoT Chemiluminescence HRP Substrate

Western BLoT Quant HRP Substrate

Western BLoT Hyper HRP Substrate

Western BLoT Ultra Sensitive HRP Substrate

（Cat. #T7101A）

（Cat. #T7102A）

（Cat. #T7103A）

（Cat. #T7104A）

II. Components (7 ml

＊

, for 70 cm

2

of membrane)

Western BLoT Chemiluminescence Luminol/Enhancer Solution

Western BLoT Chemiluminescence Peroxide Reagent

3.5 ml（light-proof bottle）

3.5 ml（clear bottle）

＊: Sample product volume

Western BLoT Chemiluminescence HRP Substrate（Cat. #T7101A）contains

each 125 ml bottle.（250 ml, for 2,500 cm

2

of

membrane

）

III. Storage

4℃

＊ Store at the recommended temperature and use within one year.

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Western BLoT Chemiluminescence

HRP Substrate Sample

IV. Materials Required but not Provided

Cat. #T7101Q

v201211

1. Protein blotted membrane:

After separating the protein sample(s) by electrophoresis, transfer proteins to a

membrane by Western blotting. If the protein-blotted membrane is to be stored, soak

in TBS-T or PBS-T (TBS or PBS containing 0.05% Tween® 20) and store at 4℃.

Either nitrocellulose membranes or PVDF membranes can be used following the

manufacturer's protocol.

2. Dilution buffer and wash buffer:

Use TBS or PBS as dilution buffer, and use TBS-T or PBS-T as wash buffer.

These can be prepared using the following products:

・TBS : Tris Buffered Saline (TBS) Tablets, pH7.6（Cat. #T9141)

・PBS : Phosphate Buffered Saline (PBS) Tablets, pH7.4 (Cat. #T9181)

Phosphate Buffered Saline (PBS) Tablets without Potassium, pH7.4 (Cat.

#T9182)

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

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

#T9183)

3. Blocking buffer:

Dissolve the blocking reagent in the same type of buffer used for antibody dilution

with Tween® 20 (i.e., TBS-T or PBS-T).

Examples of blocking reagents:

・Nonfat dry milk: recommended concentration 1 - 5%

Start with 5% as a default concenration and adjust as needed. Nonfat dry milk is

the most commonly used blocking reagent, but it interferes with the detection of

phosphorylated proteins.

・BSA: 0.3 - 3%

・Gelatin: 2%

Gelatin is not a suitable blocking reagent when using biotinylated secondary

antibodies.

Note: For additional recommendations regarding blocking buffers, please refer

to V. Precautions.

4. Primary antibody:

Choose an antibody that is specific to the target protein. Prepare a stock solution

of this antibody in dilution buffer (e.g. 1 mg/ml). Use blocking buffer to make all

working dilutions. Prepare the primary antibody working dilution. The appropriate

dilution depends on the specific primary antibody and the amount of antigen on the

membrane and requires optimization for each experimental system.

5. HRP-conjugated secondary antibody:

Prepare a 1 mg/ml stock solution of a HRP-conjugate that specifically binds the

primary antibody. Use the blocking buffer for all dilutions. Prepare a working dilution.

The optimal dilution varies depending on the HRP-conjugate and the amount of

antigen on the membrane.

6. X-ray film, film cassettes, developing solution, fixing solution, etc. required for

chemiluminescence detection.

7. Tray (large enough to hold the membrane)

8. Platform shaker: Use to agitate the membrane during the antibody reaction, washing, etc

9. Plastic wrap

10. Plastic tweezers

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Western BLoT Chemiluminescence

HRP Substrate Sample

V. Precautions

Cat. #T7101Q

v201211

These are precautions when using this product. Please be sure to review prior to use.

1. It is important to optimize the amount of sample, concentrations of primary antibody

and secondary antibody, and membrane type, buffer, and blocking reagent to be used

for each experiment.

2. The concentration of antibody required for chemiluminescence method is much less

than is required for the colorimetric method. Please run preliminary tests and optimize

the antibody concentration.

3. No blocking reagent is optimal for all systems. Therefore, it is essential to determine

the appropriate blocking buffer for each Western blot system. Determining the proper

blocking buffer can increase sensitivity and prevent non-specific signal caused by

cross-reactivity between the antibody and the blocking reagent.

4. Use adequate volumes of wash buffer, blocking buffer, antibody solution, and

Western BLoT Working Solution to sufficiently cover the membrane and ensure that

the membrane does not dry out. Using adequate quantities of blocking buffer and

wash buffer can reduce non-specific signal.

5. Non-fat dry milk can be used as blocking buffer with the Western BLoT

Chemiluminescence HRP Substrate.

Caution: Since non-fat dry milk contains biotin and phosphorylated proteins,

it cannot be used in avidin-biotin detection systems or for detecting

phosphorylated proteins.

6. To obtain the best results, please agitate the membrane throughout each incubation

step using a shaker.

7. To reduce non-specific signal, add Tween® 20 at a final concentration of 0.05% in

blocking buffer and all antibody dilution solutions.

8. Do not use sodium azide as a preservative for buffers. Sodium azide is an HRP inhibitor

and will inhibit the reaction in this system.

9. Do not handle the membrane with bare hands. Always wear powder-free gloves

or use plastic tweezers. All containers and other materials used throughout the

procedure should be free of dirt or foreign matter. Metal apparatuses, such as scissors,

should be free of rust. Rust may cause signal irregularity or high background.

10. Western BLoT Working Solution is stable for several hours at room temperature, but

exposure to direct sunlight or strong light may cause degradation. Shield the Working

Solution from light by placing it in a light-proof bottle or wrapping it in aluminum foil,

and use it as quickly as possible after preparation.

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Western BLoT Chemiluminescence

HRP Substrate Sample

VI. Protocol

Cat. #T7101Q

v201211

1. Blocking:

Prepare blocking buffer in a tray (0.5 ml for every 1 cm

2

of membrane). Remove

membrane from the Western blotting apparatus and soak in blocking buffer. Shake for

1 hour at room temperature. Discard the blocking buffer and wash twice with an

adequate quantity of fresh wash buffer.

2. Primary antibody incubation:

Dilute primary antibody to an appropriate concentration with blocking buffer.

Recommended antibody concentrations for use with this kit are as follows.

Recommended dilution ratios for stock antibody solution (1 mg/ml)

Primary antibody 1 : 1,000 - 1 : 30,000

Secondary antibody 1 : 10,000 - 1 : 100,000

Incubate the membrane in primary antibody solution diluted with blocking buffer

and shake for 1 hour at room temperature. Rinse the membrane breifly twice with an

adequate quantity of fresh wash buffer. Then, add fresh wash buffer and shake at room

temperature according to the following washing conditions:

15 minutes (use 0.7 ml of wash buffer per 1 cm

2

of membrane)

↓

5 minutes x 3 times (use 0.3 ml of wash buffer per 1 cm

2

of membrane)

3. HRP-labeled secondary antibody:

Soak the membrane in secondary antibody diluted with blocking buffer and shake for

1 hour at room temperature. Rinse the membrane breifly twice with an adequate quantity

of fresh wash buffer.

Wash with fresh wash buffer at room temperature according to the following washing

conditions:

5 minutes x 3 times (use 0.3 - 0.7 ml of wash buffer per 1 cm

2

of membrane)

4. Detection:

Bring this kit to room temperature prior to use. Prepare Working Solution by mixing equal

volumes of Luminol/Enhancer Solution and Peroxide Reagent. Use 0.1 ml of Working

Solution for every 1 cm

2

of membrane. For an 8 cm x 10 cm membrane, mix 4 ml of

each reagent to prepare a total of 8 ml of Working Solution. Prepare Working Solution

immediately prior to use.

Remove excess buffer from the membrane.

Place the membrane horizontally on a piece of plastic wrap so that the surface of the blot

faces upward. Apply Working Solution so that it is completely covers the surface.

↓

Let stand for 2 minutes at room temperature.

↓

Pick up the membrane using tweezers and remove excess Working Solution by applying

a laboratory tissue to the edge of the membrane.

Wrap the membrane in fresh plastic wrap. Make sure only one layer of plastic wrap covers

the blotted surface of the membrane. Avoid introducing air bubbles or forming creases.

↓

Detect chemiluminescent signal.

Detection using X-ray film:

Set the membrane on a film cassette so that the surface of the blot faces upward.

Place X-ray film above the blot surface and close the cassette. We recommend

three initial trial exposure times of 30 seconds, 2 minutes, and 5 minutes. Adjust the

exposure time as needed to obtain the best results. Develop the X-ray film.

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Western BLoT Chemiluminescence

HRP Substrate Sample

VII. Troubleshooting

Cat. #T7101Q

v201211

Since Western blotting comprises several steps, it may be necessary to optimize conditions.

We recommend preliminary investigations to determine the appropriate quantity of protein

for electrophoresis, the optimum dilution ratios for primary antibody and secondary antibody,

and other parameters.

Problem

High background

CauseSolution

The concentration of primary antibody Increase the dilution factor of the

used is too y antibody to decrease the

antibody concentration.

The concentration of HRP used is too Increase the dilution factor of the

ary antibody to decrease the

HRP concentration.

Too much antigen has been the quantity of antigen .

Blocking is ze blocking conditions.

The blocking reagent is a different type of blocking reagent.

Exposure time is too n the exposure time.

Washing is se the washing time, the

number of washes, or the volume of

wash buffer.

The primary antibody is m that the primary antibody

recognizes the target protein, and

the protein is not degraded.

The type of secondary antibody is Confirm that the secondary antibody

izes the primary antibody and is

not degraded.

Quantities of antigen or antibody are Increase the quantity of antigen or

dy.

Transfer of protein is ze transfer conditions.

The film exposure time is se the exposure time.

Reduced activity of HRP or substrate Use fresh reagents.

has e Working Solution

immediately prior to detection and

use within a few hours.

Efficiency of protein transfer is ze transfer conditions.

Membrane hydration is uneven.

Air bubbles have formed between the

gel and membrane.

Air bubbles have formed between the

film and membrane.

Secondary antibody has aggregated.

Particles (e.g., dust) are contaminating

the blocking buffer and/or wash

buffer.

Hydrate membrane properly.

Remove all air bubbles before

starting the transfer process.

Remove all air bubbles prior to

exposure.

Filter the secondary antibody.

Filter the blocking buffer and/or

wash buffer.

Perform experiments in a clean

environment and cover the container

with a lid during incubation and

wash steps.

The gloves being used are powder-free gloves or

change the type of gloves (nitrile,

polyethylene, etc.).

The concentration of HRP used is too Increase the dilution factor of the

ary antibody to decrease the

HRP concentration.

Reduce the concentration of HRP.

The concentration of primary antibody Increase the dilution factor of the

used is too y antibody to decrease the

antibody concentration.

No band is visible or signal is

weak

White dots are present in the

band

Irregular background

Membrane emits signal

visible to the unaided eye in

darkness; Brown bands are

present on the membrane;

An inverse image forms

Non-specific bands appear

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Western BLoT Chemiluminescence

HRP Substrate Sample

VIII. Related Products

Western BLoT Quant HRP Substrate

Western BLoT Hyper HRP Substrate

Western BLoT Ultra Sensitive HRP Substrate

Western BLoT Immuno Booster

Western BLoT Rapid Detect

Cat. #T7101Q

v201211

（Cat. #T7102A）

（Cat. #T7103A）

（Cat. #T7104A）

（Cat. #T7111A）

（Cat. #T7121A）

(Cat. #T900)

(Cat. #T903)

（Cat. #T9101)

（Cat. #T9102)

（Cat. #T9141)

（Cat. #T9142)

（Cat. #T9181)

（Cat. #T9182)

（Cat. #T9183)

PBS（Phosphate Buffered Salts）Tablets

TBS（Tris-Buffered Saline）powder

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

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

Tris Buffered Saline (TBS) Tablets, pH7.6

Tris Buffered Saline with Tween®20 (TBS-T) Tablets, pH7.6

Phosphate Buffered Saline (PBS) Tablets, pH7.4

Phosphate Buffered Saline (PBS) Tablets without Potassium, pH7.4

Phosphate Buffered Saline with Tween®20 (PBS-T) Tablets, pH7.4

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 77 543 7247 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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