ECL Western Blotting Detection Substrate. -...
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Order & Inquiry Tel: (713)732-2181 Fax: +1-866-747-4781 E-mail: [email protected] Order & Inquiry Tel: +49-89-46148500 Fax: +49-89-461485022 E-mail: [email protected] ECL Western Blotting Detection Substrate Description Note Trouble-Shooting 1. This substrate should not be in the light for a long time, please protect it from light, and manipulate in dark room. 2. In order to obtain the best result, it is necessary to optimize the concentration of primary and second antibodies. 3. Select a high quality preservative film. 4. Avoid using NaN3 to recycle HRP-conjugated antibodies, because NaN3 may inhibit the activity of HRP. 5. The solution has no toxicity. If exposed to skin, just rinse it off with running water. The Biotool ECL Western Blotting Substrate was designed for the detection of horseradish peroxidase (HRP)-labelled proteins or nucleic acids. In the chemiluminescence western blotting detection, the sensitivity was not a limiting factor due to the use of luminous enhancer. Instead, the stability and reliability, such as avoiding non-specific bands or background bands or quenching, become the key elements for successful western blotting. 1. Uses accurate luminous substrate to effectively reduce non- specific bands and background bands. 2. Contains unique luminous enhancer to detect weak signals. 3. Uses a special luminous recipe to laminate quickly and stably. 4. Not interfere with proteins on the membrane. After exposure, the blot can be stripped and re-probed several times. Components Advantages Store at 4℃ from light for 12 months. Storage Protocol 1.Perform gel electrophoresis, transferring, blocking, incubation of HRP-conjugated antibodies or nucleic acids, and wash membrane. * Recommended antibody concentration: 2.Make fresh luminous solution. Mix Solution A and Solution B at 1:1 (0.125 mL solution for 1 cm2 membrane), and transfer the final solution to clear plastic box. 3.Remove the membrane from washing solution using tweezers, and let the bottom of the membrane contact the filter paper to remove washing solution thoroughly but not dry. Immerse the membrane in fresh luminous solution, and shake gently for incubation ranging from a few seconds to one minute. 4.Remove the membrane from luminous solution using tweezers, and let the bottom of the membrane contact the filter paper to remove excess luminous solution. Quickly wrap the membrane with a clear plastic sheet protector gently smooth out any bubbles and flatten the blot. A plastic wrap also works well. Put the blot into film cassette with upward of protein and take it to dark room. 5.In dark room, put imaging film on top of the membrane, expose the film for several seconds, then develop. Repeat the exposure, varying the time as needed for optimal detection. 6.If detecting other proteins on this membrane, just wash the membrane with PBST or TBST (10 min x 3 times), and then incubate with primary antibody again. Cat B18005 size 500 mL (Solution A 250 mL + Solution B 250 mL) Primary antibody (1mg/ml) Secondary antibody (1mg/ml) 1:1,000–1:5,000, ie 0.2-1.0μg/ml 1:2,000--1:10,000, ie 0.1-0.5μg/ml Problem Possible Cause Solution White bands with a black background Membrane has brown or yellow bands Blot glows in the darkroom Weak or no signal or signal fades quickly High background Dilute HRP-conjugate further Too much HRP in the system Too much HRP exhausted the substrate Used insufficient quantities of antigen or antibodies Insufficient protein-transfer Low HRP activity Too much HRP in the system Inadequate blocking or used inappropriate blocking reagent Dilute HRP-conjugate further Optimize blocking conditions Optimize transfer conditions To test system activity, in a darkroom, prepare 1-2mL of the substrate working solution in a clear test tube. With the lights turned off, add 1μL of undiluted HRP-conjugate to the working solution. The solution should immediately emit a blue light that fades during the next several minutes. Dilute HRP-conjugate further Strip and re-probe blot using increased amount of antibodies
Transcript of ECL Western Blotting Detection Substrate. -...
Order & InquiryTel: (713)732-2181 Fax: +1-866-747-4781E-mail: [email protected]
Order & InquiryTel: +49-89-46148500 Fax: +49-89-461485022E-mail: [email protected]
ECL Western Blotting Detection Substrate
Description
Note
Trouble-Shooting
1. This substrate should not be in the light for a long time, please protect it from
light, and manipulate in dark room.
2. In order to obtain the best result, it is necessary to optimize the concentration of
primary and second antibodies.
3. Select a high quality preservative film.
4. Avoid using NaN3 to recycle HRP-conjugated antibodies, because NaN3 may
inhibit the activity of HRP.
5. The solution has no toxicity. If exposed to skin, just rinse it off with running water.
The Biotool ECL Western Blotting Substrate was designed for the detection of horseradish peroxidase (HRP)-labelled proteins or nucleic acids. In the chemiluminescence western blotting detection, the sensitivity was not a limiting factor due to the use of luminous enhancer. Instead, the stability and reliability, such as avoiding non-specific bands or background bands or quenching, become the key elements for successful western blotting.
1. Uses accurate luminous substrate to effectively reduce non-specific bands and background bands.2. Contains unique luminous enhancer to detect weak signals.3. Uses a special luminous recipe to laminate quickly and stably.4. Not interfere with proteins on the membrane. After exposure, the blot can be stripped and re-probed several times.
Components
Advantages
Store at 4℃ from light for 12 months. Storage
Protocol1.Perform gel electrophoresis, transferring, blocking, incubation of HRP-conjugated antibodies or nucleic acids, and wash membrane. * Recommended antibody concentration:
2.Make fresh luminous solution. Mix Solution A and Solution B at 1:1 (0.125 mL solution for 1 cm2 membrane), and transfer the final solution to clear plastic box.3.Remove the membrane from washing solution using tweezers, and let the bottom of the membrane contact the filter paper to remove washing solution thoroughly but not dry. Immerse the membrane in fresh luminous solution, and shake gently for incubation ranging from a few seconds to one minute.4.Remove the membrane from luminous solution using tweezers, and let the bottom of the membrane contact the filter paper to remove excess luminous solution. Quickly wrap the membrane with a clear plastic sheet protector gently smooth out any bubbles and flatten the blot. A plastic wrap also works well. Put the blot into film cassette with upward of protein and take it to dark room.5.In dark room, put imaging film on top of the membrane, expose the film for several seconds, then develop. Repeat the exposure, varying the time as needed for optimal detection.
6.If detecting other proteins on this membrane, just wash the membrane with PBST or TBST (10 min x 3 times), and then incubate with primary antibody again.
Cat B18005
size 500 mL (Solution A 250 mL + Solution B 250 mL)
Primary antibody (1mg/ml) Secondary antibody (1mg/ml)
1:1,000–1:5,000, ie 0.2-1.0µg/ml 1:2,000--1:10,000, ie 0.1-0.5µg/ml
Problem Possible Cause Solution
White bands with a black
background
Membrane has brown or
yellow bands
Blot glows in the darkroom
Weak or no signal or signal
fades quickly
High background
Dilute HRP-conjugate furtherToo much HRP in the system
Too much HRP exhausted
the substrate
Used insufficient quantities of
antigen or antibodies
Insufficient protein-transfer
Low HRP activity
Too much HRP in the system
Inadequate blocking or used inappropriate
blocking reagent
Dilute HRP-conjugate further
Optimize blocking conditions
Optimize transfer conditions
To test system activity, in a darkroom, prepare 1-2mL of the substrate
working solution in a clear test tube. With the lights turned off, add 1µL of undiluted HRP-conjugate to the
working solution. The solution should immediately emit a blue light that
fades during the next several minutes.
Dilute HRP-conjugate further
Strip and re-probe blot using increased amount of antibodies
Order & InquiryTel: (713)732-2181 Fax: +1-866-747-4781E-mail: [email protected]
Order & InquiryTel: +49-89-46148500 Fax: +49-89-461485022E-mail: [email protected]
Problem Possible Cause Solution
High backgroundUsed too much antigen
and/or antibody
Poor antibody specificity Select high quality antibody
Optimize protein transfer procedure
Hydrate membrane according to manual
Remove all bubbles before exposing blot to film
Strip and re-probe blot using decreased amount of antibodies
Spots with the protein bands
Inefficient protein transfer
Unevenly hydrated membrane
Bubble between X-ray film and membrane
Nonspecific bands
Too much HRP-conjugate
SDS caused nonspecific
binding to protein
Poor antibody specificity Select high quality antibody
Do not use SDS during immunoassay procedure
Strip and re-probe blot using a more dilute HRP-conjugate
Increase duration, time and volume of washes
Inadequate washing
Overexposed film Decrease exposure time
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