Week 13 (Biochemical Methods IV) Flashcards
Separating/analysing molecules by affinity
- Affinity chromatography
- Western blotting
- ELISA
Affinity chromatography: stationary phase
Affinity beads
Affinity chromatography: mobile phase
Liquid around the beads
Affinity chromatography matrix- Ligand on matrix :Item to be purified
(Ligand on matrix :Item to be purified)
Specific peptide sequence:Antibodies
Specific DNA sequence:Transcription factors
ATP:Protein that binds ATP
Nickel column:His-tagged protein
GSH:GST-tagged protein
Maltose:MBP-tagged protein
What is affinity chromatography matrix useful for?
- Used very frequently these days for recombinant proteins.
- Molecular biology used to add extra amino acids to the end of your protein of interest to enable easy purification. The tag can often then be cleaved off enzymatically
What are the advantages of affinity chromatography?
Easy
Excellent purification
Quick
What are the disadvantages of affinity chromatography?
Not possible for all proteins, especially those harvested from natural sources
Tags on proteins can affect expression or function
blocking- 1st step How do we visualise the protein of interest?
1st step – blocking
Membrane with transferred proteins bound The membrane loves to bind protein, need to ‘block’ all the remaining sites where proteins haven’t bound from the transfer Wash the membrane in 4% milk solution or BSA
Primary antibody- 2nd step
2nd step – 1° Antibody Need an antibody specific for your protein of interest Place the membrane in a solution containing your 1° antibody
Washing’s- 3rd step
3rd step – washing Any non-specifically bound antibody will be washed off Leaving antibody bound only where it is bound to the specific protein of interest
antibodies bind to their antigens with
very high affinity
Secondary antibody- 4th step
4th step – 2° Antibody An antibody that recognises the 1° antibody Antigen binding sites - antigen specific Constant region - animal specific ie. all antibodies produced in mice have a common sequence in this region, that differs from antibodies produced in rabbits or goats.
Washing (again) - 5th step
5th step – washing Any non-specifically bound antibody will be washed off Leaving antibody bound only where it is bound to the specific protein of interest
Developing-6th step
Place the membrane in a solution containing the substrate for the enzyme that is conjugated to the 2° antibody
Enzyme reaction will occur only at position where the 2° antibody is located
What happens If alkaline phosphatase enzyme is used in developing?
produces a purple band on the membrane wherever the protein of interest is located
What happens if horeseradish peroxidase enzyme is used in developing?
produces light.
Need to expose to film and will get a band on the film wherever the protein of interest is located
Use of Western Blotting
Protein visualisation/analysis
Advantages of western blotting?
Specificity Sensitivity (even in complex mixtures)
Disadvantages of Western blotting?
Not possible to extract the protein, only visualise Time-consuming
What is ELISA?
Enzyme-linked immunosorbant assay
What can be used to detect & quantify a protein in solution?
antibodies
what is Indirect ELISA used for?
For quantitating amount of an antibody in a sample
What are the steps in indirect ELISA? *
- There is an antigen coated well 2.Block with non specific protein 3.Add sample and if it contains antibodies they binds to antigen 4. Wash Add enzyme-linked 2° (secondary) antibody 5. Wash 6.Add enzyme substrate 7. Measure absorbance (typically turns yellow)
What Is Sandwich ELISA used for?
For quantitating amount of an antigen in a sample
What are the steps in sandwhich ELISA?
- Antibody coated well 2.Block-with non specific protein 3.Add sample 5. Antigen (If present) binds to antibody 6.Wash 7.Add enzyme-slinked second antibody 8.Wash 9.Add enzyme substrate 10.Measure absorbance
What is the main use of ELISA?
Protein detection/quantitation Detection and quantisation of small molecules
advantages of ELISA
Specificity Sensitivity (even in complex mixtures) Quantitative
Disadvantages of ELISA
Time consuming Average of all the molecules in a solution even if in different states
What does transfer involve?
Transfer from SDS page to nitrocellulose or PVDF membrane
What is Mass spectrometry?
Precise and sensitive measurement of atomic composition of a molecule without prior knowledge of its identity
What can you find out from mass spectrometry?
The mass of a protein can be precisely determined - give info on the identity and chemical state of a protein A protein can be identified from the pattern of peptide fragments formed after treatment with a specific protease A peptide can be sequenced Oligosaccharides can be sequenced Lipid species can be identified
What are the main components of mass spectrometry?
Mass spectrometers convert analyte into gaseous charged forms (gas-phase ions) A mass analyzer separates the ions according to size A detector measures the abundance of each ion
Forming gas-phase ions Two most commonly used techniques to form gas-phase ions of large biochemicals are:
MALDI (Matrix-assisted laser desorption/ionisation) ESI (Electrospray ionisation)
What is MALDI?
Sample is dried down in the presence of a volatile compound (matrix). A laser excites & vaporises the matrix converting the sample into the gas phase. Gaseous collisions cause ionisation.
What is ESI?
Sample in solution is passed through an electrically charged nozzle into a chamber of very low pressure, evaporating the solvent and giving the charged gaseous analyte
A commonly used mass analyzer is:
TOF Time of flight Gaseous ions are accelerated through an elongated chamber under a fixed electrostatic potential. A smaller ion will move through the chamber faster than a large one of the same charge.
Identification of proteins using mass spec
Identify a spot or band in a gel Cut it out Digest with trypsin Run on mass spec. Identify the protein from the pattern of peptide fragments by comparing with a database
Peptide sequencing
After going through one mass analyzer (to separate different peptides) Bombard peptide with an inert gas to break it up Enter 2nd mass analyser Identify amino acid sequence Called ‘tandem mass spec.’ or ms/ms
Post-translational modifications
Post-translational modifications are critical to the function of many proteins eg. Glycosylation Phosphorylation
Phosphorylation will increase the mass of the peptide that is phosphorylated – what can mass spectrometry detect?
amount and position of phosphorylation
What would happen if your sample did not contain any antigen?
There would be no second antibody binding therefore no signal would be produced
Which test is ELISA commonly used in?
HIV
start with an SDS page
western transfer
