Protein methods

Protein characterization includes biochemical and biophysical methods for determining the properties of a protein or for displaying a proteome.

Properties

Proteins are usually released to evaluate their properties by cell lysis and separated by means of a protein purification from the other constituents of the starting material.

Identification

The amino acid sequence can be determined by Edman degradation or mass spectrometry by a de novo sequencing by in-gel digestion. Post-translational modifications can be determined by mass spectrometry via Western blot or.

The identification of a protein can also be effected using specific antibodies by immunolabeling in a Western blot or by affinity chromatography. Similarly, a proof must be supplied also by measuring its function in enzymes by enzyme kinetics. Clearly identifiable sections of a protein can be detected by in-gel digestion by mass spectrometry ( MALDI -TOF, ESI-MS/MS, by LC-MS/MS), a peptide mass fingerprint or a de novo sequencing, then about databases as Mascot identified.

Transposon tagging, TILLING, or the untargeted mutagenesis permits the identification of protein-coding genes from the change in phenotype followed by determination of the DNA sequence of changing the phenotype of the mutation.

Size and mass

A determination of the molecular weight of a protein can be determined by mass spectrometry, for example, by gel permeation chromatography, by SDS-PAGE, by isopycnic centrifugation by field flow fractionation, or.

Form

Protein folding is, for example, by X-ray diffraction (XRD ), by cryo- EM or determined by NMR. Changes in protein folding can be followed via FTIR spectroscopy or by Circulardichroismusmessung.

Protein folding can be partially confined in the course of a protection assays by limited proteolysis or thermal denaturation with protein dyes. The surface of a protein can be determined by a deuterium exchange near-surface hydrogen atoms.

Function

Proteins can bind other molecules with the affinity each corresponding enzymes in addition comes before a catalytic activity.

Interaction

In addition, protein -protein interactions and interactions with other molecules, for example, by affinity chromatography, co- immunoprecipitation, spine and other pull-down assays, molecular display, yeast two -hybrid system, chemical crosslinking and SDS-PAGE or mass spectrometry, Native gel electrophoresis, Far - Western blotting, ligand binding assays ( radioligand, reporter enzymes, fluorescent ligands, electric discharge ), label transfer, proximity ligation assay, affinity electrophoresis, alanine scan, microscale thermophoresis, isothermal titration calorimetry, surface plasmon resonance spectroscopy, bio -layer interferometry, fluorescence correlation spectroscopy, Förster resonance energy transfer, bioluminescence resonance energy transfer, bimolecular fluorescence complementation, protein fragment complementation, density gradient centrifugation or gel permeation chromatography determined.

Similarly, a gene knockout lead to cellular and phenotypic effects that provide an indication of the function of a protein. By comparing the amino acid sequence databases such as BLAST and Pfam sequence-related proteins can give an indication of the possible functions of the protein to be tested with known functions.

Methods for the determination of protein -DNA interactions ( DNA sequence of DNA -binding proteins ) are, for example, EMSA chip DAMiD, chip-on- chip or chip -Seq. Protein-RNA interactions can be detected, for example by RNA -Seq, RIP - chip, iCLIP, CLIP -Seq or by PAR -CLIP.

Catalysis

Protein with unknown function, such as enzymatic catalysis properties are investigated by determination of enzyme kinetics. These inhibitors are often different to the function of proteins involved may be used. An alanine scan can be used to determine the active site.

Amount

Determination of the amount (quantification ) of a protein is, for example, by photometric, ELISA, SDS -PAGE and Western blot, 2D - PAGE, or by mass spectrometry ( iTRAQ, SILAC, ICAT, the Isobarenmarkierung Tandem Mass Tags ).

By photometry, the protein concentration can also be determined by the absorption of the peptide bond, for example, by Bradford assay, by Lowry assay, by biuret reaction by BCA assay or at higher protein concentrations, each method has its own interfering substances, which in the presence of a Use this method to exclude.

Various dyes preferentially bind proteins, such as Coomassie Brilliant Blue, Fast Green FCF, amido black or Ponceau S. The fluorescent dye Nile Red, for example, scopoletin, iridium complexes, SYPRO Orange, SYPRO Red, SYPRO Ruby, SYPRO be Tangerine, Flamingo, Coomassie Fluor Orange, Lucy 506 Lucy 565 Lucy 569 or Epicocconon ( Lightning Fast, Deep Purple Stain ) was used.

By peptide binding proteins ultraviolet light absorbing at a wavelength of about 205 nm (190 nm to 230 nm), in addition also absorb phenylalanine, tyrosine and tryptophan UV light at wavelengths of 280 nm to 288 nm This absorption can be used for photometric quantification and determination of the purification factors are used.

Modification

Unlike carbohydrates and nucleic acids, some structural motifs come through the various amino acids contained only proteins before, such as sulfhydryl -containing Cysteine ​​or phenolic residues in tyrosines. These can be selectively labeled with appropriate reagents. Some protease inhibitors lead to a selective labeling of proteins. Fixative usually lead to cross-linking of various amino acids.

Cleavage

By cyanogen bromide, a protein can be chemically cleaved at methionine residues contained, from the methionine produced the homoserine lactone. Isothiocyanates such as PITC with some N- terminal amino acid can be eliminated, which can also be applied in the course of the Edman degradation repeated. Selective cleavages can be enzymatically effected by endopeptidases that require longer recognition sequences compared to non-enzymatic cleavages.

Expression patterns

The different protein expression in a proteome, for example, by gel electrophoresis (2D -PAGE), protein arrays, Difference Gel Electrophoresis, MeCAT or ICAT are presented.

Electrospray ionization Peptide mass fingerprinting Mascot (Software) Electron microscope Protein–protein interaction Far-western blotting Bio-Layer Interferometry DNA-binding protein Electrophoretic Mobility Shift Assay RNA-Seq RIP-Chip PAR-CLIP Alanine scanning Isobaric tag for relative and absolute quantitation Stable isotope labeling by amino acids in cell culture Isotope-coded affinity tag Isotopic labeling Bradford protein assay Amido black 10B Digital Object Identifier Electrophoresis (journal)
662627
de