Proteins Flashcards
How can we make more proteins than the 20000 genes which our human genome consists of ?
Because the genes can be spliced differently to produce different proteins
Also can chemically modify proteins at particular residues
How much of the dry weight of an organism is protein ?
50%
What residues in proteins can be phosphorylated ?
Ones which have a hydroxyl side chain
Eg serine-10, serine-16, tyrosine-6 and threonine-17
Aspartic acid and cysteine can also be phosphorylated
How would you determine which residue of phospholambam was phosphorylated in Vivo ?
Mouse provided with radioactive phosphate which combines with ATP
Mouse stimulated with beta agonist
Mouse is then killed at the appropriate time
Heart is removed immediately and frozen to preserve proteins
Proteins of the heart are dissolved by homogenisation in gentle non denaturants detergent
Solution also contain phopshatase inhibitors and magnesium chelates to prevent phosphorylation/dephosphorylation
Phospholambam can be separated using a specific antibody and then it can be sequenced and the sites of phosphorylation with fluoresce
Give examples when total serum protein concentration needs to be assessed ?
To look for nutritional abnormalities, kidney disease, liver failure
What is the normal levels of albumins and immunoglobulins in serum protein and what can a high or low serum protein concentration indicate ?
6-8.3g/dL or 600-830g/L
High- chronic inflammation of an infections(HIV, hepatitis c or b), multiple myeloma
Low- liver disease, malabsorption and agammaglobulinaemia
Why is total protein concentration of urine tested ?
Kidney diseSe, nephropathy in diabetes and several cardiovascular diseases
What is the Lowry assay ?
Technique for measuring total protein concentration by copper chelation by the protein
What is the Bradford assay ?
Technique for measuring total protein concentration by a dye binding assay
What amino acids absorb light in the wavelength of 280Nm and why ?
Tyrosine, tryptophan and phenylalanine
They have aromatic rings in their side chains which have delocalised electrons which can absorb the light
What is the molar extinction coefficient ?
It is a measure of how strongly a protein will absorb light at a particular wavelength
Can all proteins be detected equally with the A280 technique ?
No
Because it is determined by the frequency of tyrosine, tryptophan and phenylalanine residues in the protein
Can all proteins be detected equally using the Bradford assay ?
No
Because the dye used in this assay which is coomassie blue only binds to positively charged residues in an acidic environment
Mainly detects arginine, histidine and lysine
Can all proteins be detected equally in the Lowry assay ?
Yes
Because it is based on copper chelation of the backbone so it doesn’t rely on the different residues in a protein
What features are unique to particular proteins and can be exploited for extraction ?
Primary sequence
Ligand binding sites
Antibody binding sites
Unique spectral feature
What is the unique feature of green fluorescent protein ?
Chromophore- it autocatalyses
Electron microscopy can detect it at 509nm
What feature is unique to ryanodine receptors ?
Unique ligand binding site for ryanodine, a plant alkaloid
Used to isolate the protein
What is 2D electrophoresis ?
Separation of a protein based on isoelectric point and molecular weight
Firstly separated by isoelectric point, dissolved in non ionic detergent, showing the proteins charge based on primary sequence and this causes the protein to move towards one of the electrodes and once it has reached its isoelectric point it stops
Then SDS-PAGE is used to separate the protein based on molecular weight
How is the primary sequence used to extract a protein ?
Use mass spectrometry to asses the sequence of a protein
Analyse large numbers of proteins
How is a unique antibody site used to analyse a protein ?
Bind the antibody to the site to extract the protein
Assays used include western blot, ELISA and antibody array
What are western blots ?
Analogous to southern or northern blotting but an antibody is used instead of a nucleic probe
First it is electrophoresis to separate protein based on molecular weight
Transferred to a membrane to bind proteins which creates a print of separated proteins
Non specific binding sites are blocked
Incubated with a primary antibody to bind target protein
Allows image of the blot to be viewed and the intensity of the labelling is dependent upon the amount of protein present
What does it mean by indirect method in western blotting ?
It means a primary and a secondary antibody are used and it is the secondary antibody which is labeled and highlight the binding site
Why is the intensity of labelling only comparable on the same blot ?
Because of the inconsistencies of antibody binding
Expression of labelling on different blots is not comparable which limits the number of samples that can be analysed at once - on a gel, only 10-20 lanes
