IGEM:IMPERIAL/2009/Assays Protocols/M3 heatinduction

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Heat induction

cI repressor assays

Presence of cI repressor

1) Cells are lysed

2) cI repressor is purified from cell lysate by purification tags

3) Run SDS-PAGE and visualise using Coomassie Blue staining

4) Check if protein band is correct size (theoretical is 21kDa, although our actual cI could vary a bit)

5) Transfer to nitrocellulose overnight

6) Dyes are eluted into solution using DMSO [1]

7) Resulting solution read spectrophotometrically at 600nm.

8) Absorbance linked to protein concentration by doing a standard curve using BSA of known concentration

cI physical binding to P lambda (P5)

Essentially uses principle of affinity of cI transcription factor for P lambda promoter.

This can be done by DNA footprinting, but below the other method of microarrays are applied:

1) P5 lambda promoter is amplified using PCR

2) P5 lambda promoter DNA is attached to glass slide

3) Fluorescent tags are added to cI repressor

4) Cell is lysed and cell lysate is added to glass slide

5) Glass slide is washed and fluorescence is measured.

6) To quantify, a standard curve of fluorescence with concentration of cI is generated.

Useful Readings

  1. Goldring JP and Ravaioli L. Solubilization of protein-dye complexes on nitrocellulose to quantify proteins spectrophotometrically. Anal Biochem. 1996 Nov 15;242(2):197-201. DOI:10.1006/abio.1996.0453 | PubMed ID:8937562 | HubMed [cI1]