Method of centrifugation?
- Fractionation- e.g. ground in a blender, osmotic shock, ultra-sonication (sound waves agitate) (Detergents?) to break the PM’s and free the organelles while keeping them in tact largely.
- This is called the cell Homogenate, with cell fractions.
- Preparative ultracentrifuge rotates the broken cells at high speed to separate the contents by size and density.
- High/ Low? density organelles rotate less so settle at the bottom as a pellet e.g. mitochondria, then at a slightly higher speed, ribosomes.
- These centrifugations may be repeated several times. E.g. initially at low speed 5G, and then this supernatant spun again at high speed 100,000G.
Velocity Sedimentation?
When centrifuged the components move as a series of distinct bands each at a different rate. To stop mixing (when denser towards the bottom) a density gradient is made by adding a shallow gradient of sucrose (5 to 20% going down). When centrifuged now distinct bands of sediment are created which can be separated, by collecting as drops from the bottom after puncturing the tube.
Equilibrium Sedimentation?
Ultracentrifugation.
Allows components of the cell to be separated by Buoyant density, independent of size or shape. The bands are created by a deep sucrose gradient (20-70%), and when the density of the organelles is the same as the sucrose they stay in position, with those at the bottom of the highest buoyancy density.
Ion exchange chromatography?
Ion Exchange: separates by Charge. Generally, proteins are slightly negative, so they are attracted to the positive beads, and any cations are lost. After, increasing conc of salt are used to compete for the ionic interaction between protein and bead, separating them fraction by fraction depending on charge. The positive beads are either Cation exchange Carboxy-Methyl beads (CM) or anion exchange DEAE.
3 types of chromatography?
Ion exchange: separates by charge, negative proteins stick to positive bead
Gel filtration: sepatates by size as the small molecules go into the beads and are retarded
Affinity chromatography: separates by binding, beads with covalent bound antibody to
Gel filtration chromatography?
Porous beads are used, so the small molecules are retarded as go through the beads, whereas larger move through and excluded faster, so separated by size.
Affinity chromatography?
Affinity Chromatography: Binding. If want to separate a known enzyme for example, can make a bead with covalently bound substrate to that enzyme, so it binds are is not excluded. Or DNA oligos to a specific DNA sequence. High salt or acid is then used to separate this bound protein. This creates a very pure fraction even single pass.
What is Gel electrophoresis for?
Separating proteins by molecular weight for analysis- can tell which proteins are present when compare to known.
Method of 2D Gel electrophoresis?
2D Gel Electrophoresis:
Used to achieve greater purity for complex samples. It uses Isoelectric focussing in the first dimension, separating both by charge, and SDS PAGE separating by size in the second dimension. At certain PH proteins lose their charge (denatured) so stop running at their Isoelectric point.
SDS-PAGE gel electrophoresis?
SDS-PAGE:
- SDS- detergent molecule which denatures and unwinds the protein into sticks exposing the hydrophobic molecules hidden in the centre.
- B-Mercaptoethanol can be used to break disulphide bonds between polypeptide subunits. Boiled for two minutes in these two.
- Load proteins into well and as negative run through the Polyacrylamide gel electrophoresis (PAGE) to the Anode.
- Protein migration is proportional to the molecular weight, so separates out proteins.
- This can be used to analyse how pure a protein sample is.
Difference between gel electrophoresis 2D or SDS?
2D separates by charge and molecular weight, whereas SDS separatesd by molecular weight only
Immuno/western blotting used for?
Visualising samples e.g. after electrophoresis.
Method of western blotting?
- Transfer the proteins onto a sheet of nitrocellulose paper by driving the proteins out of the gel using a strong electric current.
- Complimentary primary antibodies are made to the protein.
- Wash off excess.
- A second antibody then hybridises with this primary, which are tagged with fluorescent dyes or radioactive isotopes, so can be visualised.
Why is a secondary antibody used to the primary antibody?
Because it is more economical, e.g. can be amplified, and it is more efficient, for example the secondary antibody can be bought that are tagged and these will bind to other antibodies such as the primary. This means you dont need to tag the specific antibodies.
What are the origins of the antibodies used in immunoblotting/western?
The primary is often mouse or rabbit. The secondary anti-rabbit or anti-mouse are often donkey, and joined to horseradish peroxidase.
How are reporter lines done?
The reporter gene e.g. GFP is electroporated into the gene under the same promoter, so whenever the gene is expressed so is this, which can be visuallised as fluorescence.
How does immunofluorescence work?
Immunofluorescence (IF) is a detection method, during which antibody binding to an antigen is visualized using a fluorophore attatched to a secondary antibody, which attatches to the primary to the antigen of interest.
Methods for purifying proteins?
- Centrifugation- separates by size and density
- Chromotography:
Ion exchange: charge
Affinity: Binding
Gel filtration: Size
Purpose of gel electrophoresis?
Can tell how pure a sample is e.g. if only one band =pure.
Can use to see protein binding if more than one band or units e.g. Light chain and heavy
Can help identify proteins/Strands by molecular weight.
What is mass spectrometry used for?
Used to identify proteins by comparing to known samples.
Also, v sensitive so can show if protein has anything bound or phosphorylated.
Estimate number of AA in a sample.
Method for mass spec?
- After Protein isolation, Trypsin may be used to cleave the protein into single peptides.
- Mass spectrometry exploits the difference in ions mass/charge ratio (m/z).
- The sample is turned into a gas in the ion source.
- The sample is separated by an electric field by this ratio in the mass analyser.
- The detector creates the peaks which can then be compared to known samples to identify.
Experimental techniques for visualising proteins? (3)
Western blotting- Plymouth= proteins e.g. after electrophoresis
Immunofluorescence
techniques for protein interractions? (8)
Pull-down method Immunoprecipitation Affinity chromotography GST affinity pull down DNA footprinting EMSA Gel electrophoresis Yeast-2-hybrid screen
Experiments to show protein interraction strength?
DNA Chromatography
A radioactivity based binding
Surface plasmon resonance
