Chloramphenicol acetyltransferase (CAT) assay experiment

Summary

By determining the expression of reporter genes, such as β-gal or CAT, in transfected cells, it can be determined whether or not the guide DNA has been integrated into the host cell Mainly used for (1) determination of gene expression (2) evaluation of gene integration (3) regulation of cells. Sourced from Animal Cell Culture: A Guide to Basic Techniques (5th Edition)

Operation method

Chloramphenicol acetyltransferase (CAT) assay experiment

Principle

The chloramphenicol acetyltransferase (CAT) reporter gene transfers acetyl groups from acetyl coenzyme A to chloramphenicol. This kit uses a fluorescence assay to detect CAT activity, which has a sensitivity close to that of isotope assays, while being extremely easy and fast to perform, and at a greatly reduced cost. It is compatible with other types of reporter gene assays and protein content assays using a universal lysis buffer.

Materials and Instruments

Cell Samples
Tris-buffer Tris trinitrotoluene (Triton) CAT dilution buffer CAT enzyme standards Scintillation solution Polypropylene scintillation cups
Microcentrifuge tubes Microcentrifuge

Move

I. Materials

Sterilized items

1. D-PBSA

2. Multi-well plates: 6 wells, 35 mm

Non-sterile items

1. Tris-buffer: Tris-HCl, 0.1 mol/L, pH 8.0

2. Tris/trinitrotoluene (Triton): Tris-HCl, 0.1 mol/L, pH 8.0, 0.1% TritonX-100, 4℃ storage

3. CAT dilution buffer: Tris-HCl, 0.1 mol/L, pH 8.0, 50% glycerol, 0.2% BSA

4. Substrate: 100% methanol containing 250 mmol/L chloramphenicol (GIBCO); dispense and store at -70℃.

^{5. 14C-CoA}: ^14C-butyrate coenzyme A (10uCi/mL; Amersham Pharmacia)

6. CAT enzyme standards: Prepare CAT dilution buffer containing 0.2, 1.0, 2.0, 4.0, and 10 U/mL standards; store at 4℃.

7. "Cocktail" scintillation solution: Econofluor (Invitrogen)

8. deionized distilled water (DDW)

9. polypropylene scintillation cup: 3.5 mL

10. microcentrifuge tube

11. microcentrifuge

12. ice bath

Method A : Collection of cells from 6-well, 35 mm culture plates

1. 24-72 h after transfection, wash the cells with D-PBSA. 2. Place the plate on ice.

2. Place the plate on ice and add 1 mL of Tris/Triton to each well. 3.

3. Freeze the plate at -70℃ for 2 h. 4.

4. Thaw the plate at 37°C and place on ice. 5.

5. Transfer the cell lysate to a microcentrifuge tube and centrifuge at maximum speed for 5 min. 6.

6. Collect the supernatant and heat at 65℃ for 10 min to inactivate CAT inhibitors. 7.

7. Collect the supernatant (cell lysate) after centrifugation at maximum speed for 3 min and store the supernatant at -70 °C.

Method B: CAT assay

8. 5 to 150 uL of cell lysate from each sample is transferred to a 3.5 mL polypropylene scintillation cup and enough 0.1 mol/L Tris is added to give a final volume of 150 uL. 9. A blank cup is set up with 150 uL of 0.1 mol/L Tris as a negative control.

9. Set up a blank cup with 150uL of 0.1mol/L Tris solution as a negative control.

10. Positive control:
(a) Take 5 scintillation cups and add 150uL of 0.1mol/LTris to each.
(b) Add 5uL of CAT standard solution to each scintillation cup and plot the CAT standard curve at 1, 5, 10, 20 and 50mU. 11.

11. To all sample cups (including controls), add 100uL of the following mixture:
(a) 84uL of ultrapure water
(a) 84uL ultrapure water (b) 10uL 0.1mol/L Tris
(c) 1uL of chloramphenicol
(d) 5 uL (50 nCi) of ^14C-CoA

12. Tighten the lid of the cup and incubate for 2 h at 37°C. 13.

13. Add 3 mL of Econofluor to all scintillation cups and tighten the lids.

14. Invert up and down to mix the ingredients in the scintillation cups. 15.

15. Incubate at room temperature for 2 h. 16.

16. Determine the number of 30s flashes on a liquid flash counter.

Caveat

1. In the amplification and preparation step of the expression plasmid, special attention should be paid to its purity (not containing RNA and chromosomal DNA) so as not to affect the transformation efficiency.

2. The best preparation method is to use lysozyme plus Triton10, and then centrifuged by chlorinated Yan gradient.

3. there must be a control group with CAT enzyme instead of cell extract to verify the feasibility of the reaction and chromatography process, or Psv:CAT and Psv. cAT expression plasmid to verify the correctness of the two steps of transformation and recombination.

4. In the preparation of cell extracts, it is best to use ultrasonic crushing method, and pay attention to check the degree of crushing before centrifugation. Freeze-thawing method is cumbersome and sometimes incomplete crushing affects the results.

5. 4mmol/L ethylphthalide coenzyme A can be taken 1.smg each time and dissolved in 500 chuan water, it is best to prepare before use, the prepared solution can be stored in a 20 ℃, but not more than ten days.

Common Problems

Successful assembly of the expression plasmid is one of the keys to this assay, and the expression DNA plasmid must have the following components: (1) prokaryotic coding sequence for cellular replication and marker genes (antimicrobial genes) for cryptic selection; (2) regulatory elements controlling eukaryotic cell transcription (enhancers, promoters); (3) control of some signals, transcriptional processing trimming signals, tailing signals for use in the expression of mammals; (4) unknown sequence of genes to be tested can replace the above transcriptional regulatory elements; (5) marker gene (chloramphenicol ethacyltransferase). mammalian expression; (4) unknown sequence of genes to be tested can replace the above transcriptional regulatory sequence; (5) marker gene (chloramphenicol acetyltransferase).

Reference:
1. Animal Cell Culture: A Guide to Basic Techniques (5th Edition);
2. Chloramphenicol Acetyltransferase Assay (CATassay) Basic Medicine and Clinical

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