Boster Immunoleader

other

Hypersensitive WB Chemiluminescent Substrate

CR0001-5

5 ml (20 x)

65 USD

CR0001-5

Hypersensitive WB Chemiluminescent Substrate

5 ml (20 x)

65 USD

Western Blotting Related Reagent

4˚C in dark for at least one year.

Our Western blot chemiluminescent substrate is an enhanced chemiluminescent substrate with high sensitivity and unique chemiluminescent system. With low background, good stability, sensitive and enhanced signal, this reagent is good for detecting direct and indirect conjugated HRP antibodies and their related antigens, it can detect the target protein sensitively. Catalyzed by HRP, this chemiluminescent substrate can be used to expose X-ray film, and do luminometer detection directly or fluorescence CCD scan.

1. Operate the routine SDS-PAGE, transfer membrane and other procedures of western blot. 2. Block the membrane: wash membrane twice, 10 minutes each with TBS-T. Completely submerge the membrane in the blocking solution (5% defatted milk), and incubate at room temperature for 1 hour with agitation. 3. Remove the blocking buffer. Incubate the membrane in diluted primary antibody at room temperature for 2 hours or at 4˚C overnight with agitation. Note: Follow the antibody manufacturer’s recommendations for best concentration. And dilute the primary antibody with blocking buffer (5% defatted milk). 4. Wash the membrane with wash buffer (TBS-T, 4x or 6x) by agitating, 3 times for 10 minutes each. Added volume of the wash buffer, and/or increase the wash times can reduce the background. Note: Before wash, pre-wash with TBS-T simply, will increase the washing effect. 5. Incubate the membrane with diluted HRP conjugated secondary antibody at room temperature for 2 hours with agitation. Note: Follow the antibody manufacturer’s recommendations for best concentration. 6. Repeat step 4 to remove the non-specific conjugation of HRP conjugated secondary antibody. Note: After incubating with HRP conjugated secondary antibody (Step 5), the membrane must be washed throughly. 7. Prepare chemiluminescent substrate working solution: add Reagent A and Reagent B, 50uι for each into 1ml distilled water and mix throughly. Volume: Completely submerge the membrane, per 10 cm2 membrane may need about 1ml working solution. 8. Put the membrane on a flat (protein side upface), add the chemiluminescent substrate working solution, and incubate for 1-5 min. Note: Observe the incubation in the dark room to judge whether do film exposure. 9. Remove blot from Working Solution and place it in a plastic membrane protector; a plastic sheet protector or plastic wrap may be used. Use an absorbent tissue to remove excess liquid and to carefully press out any bubbles from between the blot and surface of the membrane protector. 10. Carefully put a piece of X-ray film on top of the membrane, expose 5 seconds to 1 minute, develop film using appropriate developing solution and fixative immediately. Note: The exposure time may be varied to achieve optimal results. If the signal is weak, prolong the exposure time to hour. Or use a storage phosphor imaging device or a CCD Camera to record chemiluminescent images directly.

For detecting direct and indirect conjugated HRP antibodies and their related antigens