- Kit Component:
- Reagent A: Luminol and enhanced chemiluminescent reagent 5 ml. 20× Reagent B: peroxide and specially manufactured stable reagent 5 ml. 20×
- Kit Detection Method:
- Colorimetric, OD450 nm
- Printable datasheet for OKBB00065
- Reconstitution and Storage:
- Store at 4C in dark for one year
- 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
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with agitation. 3. Remove the blocking buffer. Incubate the membrane in diluted primary antibody at room temperature for 2 hours or at 4C 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, 4× or 6×) 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.
- Tips Information: