The chemiluminescent Western Blot Kit for the Detection of E. coli Host Cell Proteins (HCP) combines all necessary reagents with a rapidly proven protocol for the detection of E. coli HCP contamination in products produced by recombinant expression in E. coli host cells.
Intended Use
Use the kit for the detection of host cell protein contaminations by 1D-Western blot, 2D-Western blot or by dot blot. It can be used as a process development tool or routine quality control method to monitor the optimal removal of HCP contaminants.
After protein separation and transfer, the membrane is probed with Anti-Host Cell Proteins Antibody. The membrane bound antibody-antigen complex is detected by the addition of a secondary antibody bound to the enzyme horseradish peroxidase. The enzyme reacts with Femtomax, a special formulation of luminol, an extremely sensitive, non-radioactive substrate that emits light (chemiluminescence) and allows visualization with X-ray film or other imaging techniques, including highly sensitive CCD cameras and imaging systems.
The femtomaxes reagent (FEMTOMAX-020 or FEMTOMAX-110) is not included in this kit and must be purchased separately.
E. Coli HCP Antibody used in Chemiluminescent Western Blot Kit
The antibody contained in this kit recognizes high and low molecular weight protein fractions of E. coli. This antibody has been validated by 2D Western Blot electrophoresis to ensure the highest possible coverage of E. coli HCP. Western blot 2D electrophoresis analysis provides a more comprehensive insight into the immune coverage of host cell lysate protein components. It detects impurities that are often not visible through 1-D electrophoresis. The Anti-Host Cell Proteins antibody contained in this kit detects over 300 E.coli proteins and is suitable for Western blotting.
Validation data E. Coli HCP Antibody
Western Blot of Rabbit anti-HCP antibody. Lane 1: Molecular Weight. Lane 2: Total HCP. Load: 10µg per lane. Primary antibody: Rabbit anti-HCP cocktail at 1:50 for overnight at 4°C. Secondary antibody: Goat anti-rabbit secondary antibody at 1:10,000 for 30 min at RT. Blocking buffer: ABIN925618 (antibodies-online) for 1 hour at RT.
2D Western Blot of E. coli HCP Control Protein. Load: 35 µg Total HCP. pH: 5-8. Exposure: 2.4s. Primary antibody: Rabbit anti-HCP antibody at 1:50 for overnight at 4°C. Secondary antibody: Goat anti-rabbit secondary antibody at 1:10,000 for 30 min at RT. Blocking buffer: ABIN925618 (antibodies-online) for 1 hour at RT.
Additionally Required Material
Nearly all components required for the detection of HCP proteins by western blot are provided for your convenience in the Chemiluminescent Western Blot E. Coli HCP Detection Kit. You will need some additional equipment listed below:
- SDS-PAGE electrophoresis equipment and related materials
- Nitrocellulose, PVDF (polyvinylidene difluoride) or other membranes for protein transfer and transfer materials
- Microfuge tubes
- Rocker platform for gentle mixing during incubations
- X-ray film, cassettes and related materials or camera based imaging system
- Chemiluminescent FemtoMax™ Super Sensitive HRP Substrate for Microwell and/or Membrane (2 component
system) – FEMTOMAX-020 or FEMTOMAX-110 - Deionized water and molecular biology grade Tris base and sodium chloride
More information required?
If you would like to have more information about the Chemiluminescent Western Blot Kit for E. Coli HCP, be free to contact us via email or phone, or visit the product page at antibodies-online.com.