1蛋白质分离纯化

蛋白质分离纯化

How to detect, analyze and prepare?

Different purposes, different protocols Detective/analytical level: nanogram(ng) or picogram(pg)

Preparative level: milligram (mg)

Protein detection/analysis:

1, Hybridization: Western blot

2, Immunological Techniques: Elisa & Co-IP

3, electrophoresis

SDS PAGE, 2-D gel, CE, IEF

4, chromatography: HPLC, FPLC,

5, Spectrometry: DLS, NMR, Mass-Spect, LC-MS 6, X-ray crystallography

…

Western blot: Protein-protein

This method is dependent on the use of a high-quality antibody directed against a desired protein.

Southern blot: DNA-DNA (Edward Southern. Detection of specific sequences among DNA fragments separated by gel electrophoresis.

J Mol Biol. 1975 Nov 5;98(3):503-17)

Northern blot: DNA-RNA

SDS-PAGE (sodium dodecyl(lauryl) sulfate-polyacrylamide gel electrophoresis)

has a number of uses, which include:•Establishing protein size

•Protein identification

•Determining sample purity

•Identifying disulfide bonds

•Quantifying proteins

•Blotting applications

N-terminal protein sequencing by Edman chemistry 1.Samples can be supplied in solution or as membrane blots with more than 90% purities(μg level)

2.If the sample is not pure enough, it might be separated with SDS-PAGE or 2-D gel electrophoresis and then electrotransfered to ProBlottTM PVDF membrane

3.The length limit of the sequence obtained depends on the sample amount, the protein properties etc, ranging from 30 to 70 residues.

pI: IEF (Isoelectric focusing)

Focusing separation of species in an inhomogeneous medium (pH-gradient) according to their isoelectric points (pI).

Molecular weight:

SDS PAGE (polyacrylamide gel electrophoresis) HPLC (high pressure liquid chromatography)

MS (Mass spectrometry)

Protein concentration detection:

BSA method

Co-efficient

Yeast ORF Ygr271c-ap

MDANTLNVSFEEILKGKKLDEDSIGLTLSP DKDHEDGSQVSPTQDRKELDQVVGEDEK DDFFE

No W or Y, extinction coefficient is 0

Absorption spectra of the aromatic amino acids in the near-ultraviolet region

From D. Wetlaufer, Adv. Protein Chem. (1962) 17:303-390.

©1962 Academic Press

UV detection

What is chromatography (色谱)?

it is a broad range of physical methods used to separate and/or to analyze complex mixtures. The components to be separated are distributed between two phases: a stationary phase (固定相) bed and a mobile phase (流动相)which percolates (filters) through the stationary bed.