![]() (B) Densitometry analysis shows that the Restore stripping buffer permitted both complete signal removal and maintained nearly identical levels of detection between the initial and reprobed western blot analysis. The membrane sections were reblocked and the western blot procedure repeated as described above. Following the stripping procedure, the membrane sections were washed, incubated with substrate, and imaged. Following the initial detection, the blot was cut into three sections and stripped according to the manufacturer’s instructions in either Restore Western Blot Stripping Buffer, Reblot Plus Stripping Solution, or Revitablot Western Blot Stripping Buffer. The membrane was probed with anti-Hsp90 polyclonal antibody followed by goat anti-rabbit horseradish peroxidase conjugate and imaged. (A) After blocking, the membrane was analyzed by western blot using SuperSignal West Dura Extended Duration Substrate. Three different concentrations of HeLa cell lysate were separated by SDS-PAGE and transferred to a to 0.45 μm nitrocellulose membrane. Comparison of stripping buffers on nitrocellulose. The bond between streptavidin and biotin is very strong and can be difficult to dissociate for reprobing. Minimize these effects by probing for low-abundance proteins or with low-affinity antibodies first. With every round of stripping, there is a potential for antigen loss. Use more stringent conditions if required to break up strong antigen-antibody pairs. Start with gentle, mild conditions to avoid loss of sample. Strip and reprobe only chemiluminescent and fluorescent western blotsĬolorimetric/chromogenic detection reagents leave a permanent visible stain on the membrane that will not be removed by stripping procedures. PVDF is less brittle and fragile than nitrocellulose, making it more useful when requiring multiple rounds of reprocessing (stripping and reprobing procedures). ![]() PVDF membranes have a protein binding capacity of 170–200 µg/cm 2 and offer better retention of adsorbed proteins than other supports because of the greater hydrophobicity. By stripping the membrane, the blot can be reused. Immunoblotting requires many steps, providing ample opportunity for mistakes to occur. When immunoblot results are not as expected, reprobing allows the use of the same protein sample without going back to gel electrophoresis. Pierce Protein Methods Chemiluminescent detection for western blotting is popular because it offers several advantages over other detection methods. Optimization is achieved easily by stripping the membrane and reprobing with a different antibody concentration. The light emission intensity of high sensitivity chemiluminescent substrates often require antibody concentration optimization to achieve the highest quality blot. By using the same blot for several different detections, you save time.īy reusing the same blot, you save money on the costs of the gel, membrane, and protein sample. It is time-consuming to run an SDS-polyacrylamide gel and then transfer the proteins to a membrane. ![]() Pierce ECL Western Blotting Substrate provides performance identical to the original Amersham ECL Western Blotting Detection Reagent from GE Healthcare.When the protein mixture is rare or valuable, reprobing conserves the sample and allows the membrane to be analyzed with the same or different antibodies. ![]() It enables the detection of picogram amounts of antigen and allows for easy detection of HRP using photographic or other imaging methods.īecause the luminol and peroxide reagent formulations are identical to other commercially available substrate products, one can switch to Pierce ECL without needing to optimize probing conditions or incubation protocols. Pierce ECL Western Blotting Substrate provides reliability and performance equivalent to other standard ECL substrates for detection of HRP enzyme activity. PI32106 Pierce ECL Western Blotting Substrate 500mL kit PI80197 Pierce ECL 2 Western Blotting Substrate Sufficient reagent for 250cm2 of membrane Detection Reagent A: 25mL Detection Reagent B: 0. Recommended secondary antibody concentration: 1:1,000–1:15,000 dilution (0.07–1.0 µg/mL)Ĭompare and learn more about our western blot chemiluminescent substrates ›.Compatibility: nitrocellulose and PVDF membranes.Stability: 1-hr working solution stability, 1-year kit stability at 4☌.Pierce ECL Plus Western Blotting Substrate characteristics include: Thermo Scientific Pierce ECL Western Blotting Substrate is a value-priced, entry-level horseradish peroxidase (HRP) substrate for enhanced chemiluminescence (ECL) that directly replaces costlier products without the need to re-optimize conditions. Pierce ECL Plus Western lotting Substrate Required materials Western blot membrane X-ray film or CCD imaging system (e.g. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |