ecl plus substrate
kit contents: ecl plus solution a, 50 ml; ecl plus solution b, 50 ml; sufficient for 1000 cm2 of membrane
storage:upon receipt store solutions at 4°c. when stored at room temperature, substrate components are stable for six months. these products are shipped at ambient temperature.
important notice: please use the same blotting conditions when switching from thermo scientific pierce supersignal western ecl plus substrate or other similar ecl substrates. the ecl plus has been developed to be more sensitive compared to western ecl plus substrate or other similar ecl substrates. however, if you are currently using fbk femto ecl substrate (cat # f046),switching to fbk pico substrate may require increasing the antigen and antibody concentrations. careful titration of antigen and antibody concentrations is recommended.
introduction
the ecl plus substrate is a highly sensitive nonradioactive, enhanced luminol-based chemiluminescent substrate for the detection of horseradish peroxidase (hrp) on immunoblots. the ecl plus substrate enables the detection of sub-picogram amounts of antigen and allows for easy detection of hrp using photographic or other imaging methods. blots can be repeatedly exposed to x-ray film to obtain optimal results or stripped of the immunodetection reagents and re-probed. the optimally formulated ecl plus substrate makes it the ideal substitute for pierce supersignal western ecl plus substrate and other similar substrates without the need for additional optimization of assay conditions.
important product information
1. for best results, it is essential to optimize all components of the system including sample amount, primary and secondary antibody concentration, and the choice of membrane and blocking reagents. use the same blotting conditions when switching from pierce supersignal western ecl plus substrate to ecl plus substrate.
2. the antibody concentrations required are lower than those used with our competitors' similar products. to optimize the appropriate concentrations, perform a systematic dot blot analysis.
3. because no blocking reagent is optimal for all systems, empirical testing is essential to determine the appropriate blocking buffer for each western blot system. determining the proper blocking buffer can help increase sensitivity and prevent nonspecific signal caused by cross-reactivity between the antibody and the blocking reagent. furthermore, when switching from one substrate to another, a diminished signal or increased background sometimes results because the blocking buffer was not optimal for the new system.
4. avoid using milk as a blocking reagent when using avidin/biotin systems because milk contains variable amounts of endogenous biotin, which will result in high background.
5. use a sufficient volume of wash buffer, blocking buffer, antibody solution and substrate working solution to cover blot and ensure that it never becomes dry. using large blocking and wash buffer volumes may reduce nonspecific signal.
6. for optimal results, use a shaking platform during incubation steps.
7. add tween®-20 (final concentration of 0.05%) to the blocking buffer and when preparing all antibody dilutions to reduce nonspecific signal.
8. do not use sodium azide as a preservative for buffers. sodium azide is an inhibitor of hrp and could interfere with this system.
9. do not handle membrane with bare hands. always wear gloves or use clean forceps. all equipment must be clean and free of foreign material. metallic devices (e.g., scissors) must have no visible signs of rust. rust may cause speckling and/or high background.
10. the substrate working solution is stable for 4 hours at room temperature. exposure to the sun or any other intense light can harm the working solution. for best results keep the working solution in an amber bottle and avoid prolonged exposure to any intense light. short-term exposure to laboratory lighting will not harm the working solution.
procedure summary
note:therecommendedrangeofantibodydilutionsshouldbeusedasreferencestoobtainpositiveresults.theoptimalantigenandantibodyamountstobeusedmayrequireexperimentation.
1. diluteprimaryantibodyto100-500ng/ml.
2. dilutesecondaryantibodyto10-50ng/ml.
3. mixthetwo substratecomponents(aandb)ata1:1ratiotopreparethesubstrateworkingsolution.
note:exposuretothesunoranyotherintenselightcanharmtheworkingsolution.forbestresultskeeptheworkingsolutioninanamberbottleandavoidprolongedexposuretoanyintenselight.short-termexposuretolaboratorylightingwillnotharmtheworkingsolution.
4. incubateblot5minutesinecl plussubstrateworkingsolution.
5. drainexcessreagent.coverblotwithclearplasticwrap.
6. exposeblottox-rayfilmoruseimagingdevices.
additional materials required
1) completed western blot membrane: use any suitable protocol to separate proteins by electrophoresis and transfer them to a membrane.
2) dilution buffer: use either tris-buffered saline (tbs) or phosphate-buffered saline (pbs).
3) wash buffer: add 5ml of 10% tween-20 to 1000ml dilution buffer. (final concentration of tween-20 will be 0.05%.)
4) blocking reagent: add 0.5 ml of 10% tween-20 to 100ml of a blocking buffer. choose a blocking buffer with the same base component as the dilution buffer.
5) primary antibody: choose an antibody that is specific to the target protein(s). use the blocking reagent to prepare a primary antibody working dilution ranging from 100 ng/ml to 500 ng/ml. for example, if the primary antibody is supplied at 1 mg/ml, dilute it in the range from 1:2,000 to 1:10,000. the necessary dilution to use depends on the specific primary antibody and the amount of antigen on the membrane and will require optimization for each experimental system.
6) secondary antibody: use the blocking reagent to prepare a hrp-conjugate working dilution ranging from 10 ng/ml to 50 ng/ml. for example, if the antibody is supplied at 1mg/ml, dilute it in the range from 1:20,000 to 1:100,000. the necessary dilution varies depending on the primary antibody, hrp-conjugate and amount of antigen on the membrane
7) and will require optimization for each experimental system.
8) film cassette, developing and fixing reagents: for processing autoradiographic film. or
9) imaging devices: e.g., bio-rad’s molecular imager system or a similar gel documentation system.
10)rotary platform shaker: for agitation of membrane during incubations.
| name | ecl plus | ||
|---|---|---|---|
| cat# | 044-100ml; 044-500ml | cas# | n/a |
| storage# | 4°c干燥避光 | shelf life# | 12个月 |
| ex(nm)# | n/a | em(nm)# | n/a |
| mw# | n/a | solvent# | n/a |
| name | ecl plus |
|---|---|
| cat# | 044-100ml; 044-500ml |
| cas# | n/a |
| storage# | 4°c干燥避光 |
| shelf life# | 12个月 |
| ex(nm)# | n/a |
| em(nm)# | n/a |
| mw# | n/a |
| solvent# | n/a |