candyswift | creativebiolabs
Protein Expression, Isolation and Purification
Dec 29th 2015 at 1:33 AM
- Learn the approaches and meaning of the expression of cloned genes.
- Learn the method of recombinant protein affinity chromatography, isolation and purification
- LB fluid medium: Tryptone 10g, yeast extract 5g, NaCl 10g.Mix all together with distilled water to 1000mL.
- Ampicillin: 100mg / mL
- Sample: Buffer Solution. 100 mM NaH2PO4, 10 mMTris, 8M Urea, 10 mM2-ME, pH8.0
- Washing Buffer: 100 mM NaH2PO4, 10 mM Tris, 8 M Urea, pH6.3
- Elution Buffer: 100mM NaH2PO4, 10 mMTris, 8M Urea, 500 mM Imidazole, pH8.0
- Inoculate BL21 E. coli strain containing recombinant chloramphenicol acyltransferase protein in 5mL LB liquid medium (containing 100ug / mL ampicillin), Grow liquid cultures with agitation in shaking incubator overnight at 37 ℃.
- Transfer 1mL of this culture to 100mL LB liquid medium (containing 100 ug / mL ampicillin) and treat the mixture with shaking incubator until OD600 = 0.6 – 0.8. Take 10ul of the sample for SDS-PAGE analysis.
- Add IPTG to make a final concentration of 0.5 mmol / l and then continue to culture at 37 ℃ for 1-3h.
- Centrifuge for 10min at 12,000rpm. Discard the supernatant. Keep the insoluble material at -20 ℃ or -70 ℃.
- Preparation of NTA chromatography column: add 1mL NTA into the chromatography column media, wash with 8mL of deionized water and 8mL of buffer elution respectively.
- Denaturing of recombinant protein: thaw the insoluble material in ice bath, add 5mL buffer elution in it, resuspended with a straw, vortex the bacteria with ultrasonic. Mix gently the sample, centrifuge at 12000rpm for 60min at 4 ℃, and collect supernatant Take 10ul for SDS-PAGE analysis.
- Pour the supernatant onto the Ni2 + -NTA column at 10-15mL / h of flow rate. Collect the liquid flowed out and take 10ul for SDS-PAGE analysis.
- Elution of impure protein: wash the column at 10-15mL / h with Washing Buffer till OD280 = 0.01. Repeat elution two to three times and analyze each fraction by SDS-PAGE.
- Elution of the target protein: wash by adding Elution Buffer. Take 10ul of each sample respectively for SDS-PAGE analysis.
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