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11363727001

Roche

CAT ELISA

sufficient for 192 tests, kit of 1 (10 components), suitable for ELISA

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Synonym(s):
chloramphenicol acetyltransferase

usage

sufficient for 192 tests

packaging

kit of 1 (10 components)

manufacturer/tradename

Roche

technique(s)

ELISA: suitable

storage temp.

2-8°C

General description

Promoter activity in transfected eukaryotic cells is generally studied by linking the promoter sequence to a gene that encodes an easily detectable “reporter” protein, such as chloramphenicol acetyltransferase (CAT), β-galactosidase (β-Gal), or luciferase (Luc).
The bacterial enzyme chloramphenicol acetyltransferase (CAT, type I), encoded by Tn9 and having no eukaryotic equivalent, has become one of the standard markers used in transfection experiments. Traditionally, CAT activity is measured using a radioisotopic CAT assay.
The CAT ELISA is specific for CAT (type I) (Acetyl-CoA:chloramphenicol acetyltransferase, EC 2.3.1.28) from E. coli (Tn9-encoded).

Application

For research use only. Not for use in diagnostic procedures.
The CAT ELISA is used to quantitatively measure CAT expression in eukaryotic cells that are transfected with a plasmid bearing a CAT-encoding reporter gene.

Features and Benefits

The CAT ELISA is specific for CAT (type I) (Acetyl-CoA:chloramphenicol acetyltransferase, EC 2.3.1.28) from E. coli (Tn9-encoded).
  • Standardized: Allows direct comparison of data from different sets of experiments, even when kits from different production lots are used
  • Faster: Approximately 4hours from start to finish
  • Safer: No radioisotopes are used
  • More accurate: Measures the actual amount of CAT protein synthesized, not just CAT enzyme activity
  • Sensitive: Equivalent to the sensitivity of the radioactive CAT assay
  • Easy to perform: Follows a standard ELISA protocol in microplates; allows Triton lysis of the transfected cells (CAT activity in the radioisotopic assay is rapidly inhibited by Triton)
  • More flexible: Allows the analysis of promoters in plant protoplasts (alternative method to the GUS assay) and animal cells

Packaging

1 kit containing 10 components.

Principle

The CAT ELISA is based on the sandwich ELISA principle. Following lysis of the transfected cells, the cell extracts (that contain CAT enzyme) are added to the wells of the microplate, which is precoated with a polyclonal antibody to CAT (anti-CAT). All CAT contained in the cell extracts binds to the anti-CAT antibody that is bound to the plate surface. Next, a digoxigenin-labeled antibody to CAT (anti-CAT-DIG) is added and binds to CAT. In the next step, an antibody to digoxigenin, conjugated to peroxidase (anti-DIG-POD), is added and binds to digoxigenin. In the final step, the peroxidase substrate ABTS is added. The peroxidase enzyme catalyzes the cleavage of the substrate, yielding a colored reaction product. The absorbance of the sample is determined using an ELISA reader and is directly correlated to the level of CAT present in the cell extracts. The sensitivity of the assay can be enhanced using ABTS with the substrate enhancer.

Preparation Note

Working solution: CAT enzyme working dilution
Add 40 μl CAT enzyme stock solution (solution 1) to 3.96 ml Sample buffer (solution 7) to obtain a CAT enzyme working dilution (final concentration approx. 1 ng/ml), sufficient to produce a calibration curve in duplicate.

Handling instructions

  • The CAT enzyme standard dilutions should be prepared freshly before use and should not be stored.
  • Prepare the standard dilution series in reaction tubes in 1:2 dilution steps as described in the table below.
  • To obtain a calibration curve, we recommend using the five concentrations listed.
  • 200 μl of each dilution is needed per well.
  • To ensure that the measurements and the calibration curve are accurate, we recommend preparing two samples of each concentration for measurement.
  • To avoid carryover of the higher conc. solution to the lower conc. samples, use a fresh pipette tip for each dilution step.
  • Each dilution must be measured in duplicate.

Storage conditions (working solution): Sample Buffer 2 to 8 °C
Stability of ready-to-use sample buffer: 2 months at 2 to 8 °C, otherwise aliquoted at - 15 to -25 °C.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit Components Only

Product No.
Description

  • CAT Enzyme (type I), from E. coli

  • Anti-CAT-digoxigenin antibody

  • Anti-Digoxigenin-POD antibody

  • POD Substrate

  • Substrate Enhancer

  • Washing Buffer 10x concentrated

  • Sample Buffer

  • Lysis Buffer 5x concentrated

  • Microplates, precoated with anti-CAT (2 x strip frame with 12 modules of 8 wells)

  • Self-adhesive Plate Cover Foils (3)

See All (10)

Pictograms

Exclamation markHealth hazard

Signal Word

Danger

Hazard Classifications

Aquatic Chronic 3 - Carc. 1B - Eye Irrit. 2 - Muta. 1B - Skin Sens. 1

Storage Class Code

6.1D - Non-combustible, acute toxic Cat.3 / toxic hazardous materials or hazardous materials causing chronic effects

WGK

WGK 2

Flash Point(C)

does not flash


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