Protein analysis and post-translational modifications Flashcards
(34 cards)
What are the two main techniques for analysing protein size?
-gel electrophoresis
-mass spectrometry
What does gel electrophoresis provide in terms of protein analysis?
it provides the approximate mass of proteins
What is the purpose of Mass Spectrometry in protein analysis?
provides accurate mass and can give amino acid sequences
Describe SDS-PAGE
a technique that separates denatured proteins based on their mass using Sodium Polyacrylamide Gel Electrophoresis
What is the difference between SDS-PAGE and Native PAGE?
-SDS-PAGE separates denatured proteins based solely on mass.
-Native PAGE separates proteins based on both mass and charge at a given PH.
Describe electrophoresis
the movement of molecules through a gel matrix in an applied electrical field
Which way do molecules with a negative charge move?
the anode (+ve electrode)
In SDS-PAGE, how are polypeptide chain migrations determined?
by molecular weight
-smaller chains run further
What is Isoelectric Focusing (IEF)?
a technique used to separate proteins according to their overall charge in the first dimension of 2D gel electrophoresis
What do disulphide bonds need to be reduced with in SDS-PAGE? What is this for?
with a reductant such as dithiothreitol etc. to ensure protein is fully denatured
In IEF, when PI=PH, what will happen to the movement of proteins?
the protein will stop
What could SDS be described as?
an ionic surfactant, lots of SDS binds when the protein is unfolded
What is 2D-PAGE?
a technique that combines IEF and SDS-PAGE for comprehensive protein separation analysis
Describe the role of trypsin in the analysis of protein mixtures
trypsin digests proteins extracted from gels for identification by peptide mass fingerprinting
What is Proteomics?
the study of the proteome, including the identification and characterisation of proteins in complex mixtures
What about a protein could also be known?
if a particular protein is expressed and under what physiological circumstances
Name the first step for analysing whole cells or tissue
1.) Exact mixture of protein from cells/tissue and analyse by 2D-PAGE
Name the second step for analysing whole cells or tissue
2.) Extract spots (i.e proteins) from gel, digest with trypsin
Name the third step for analysing whole cells or tissue
3.) Identify by peptide mass fingerprinting
Why is protein purification necessary?
to isolate specific proteins for study and analysis
What are Post-Translational Modifications?
chemical modifications that occur on proteins after translation, affecting their function and activity
What are some examples of covalent post-translational modifications?
-disulphide bridge formation
-cleavage of peptide bonds
-modification of N-terminus or C-terminus
What is Glycosylation?
the addition of carbohydrate (polysaccharide) moieties to proteins
-this enhances their stability
What types of glycosylation exist and what are their associated amino acids?
-N-linked glycosylation involves asparagine
-O-linked glycosylation involves serine and threonine
