Lec11/12 - Intro to Experimental Methods in Protein Biochemistry Flashcards
State the 3 general stages of protein purification
- Sample homogenisation
- Differential centrifugation to isolate different cellular compartments
- Separation of proteins based on (size, solubility, charge, etc.)
Name 5 methods of cell lysis (first step in protein purification)
Mechanical, Freeze/Thaw, Liquid Homogenisation, Sonication, Manual Grinding
State the equation for acceleration during centrifugation (and what the symbols mean)
a = w^2 x r
(w = angular velocity = 2π rpm / 60)
(r = radius in m)
What is the impact of doubling the speed or radius on the acceleration of a centrifuge?
Double radius = Double acceleration
Double speed = 4 x acceleration (because a = w^2 x r)
How are proteins separated based on solubility? (Name of principle, and brief summary)
Crude Separation: proteins are precipitated by adding competing solutes (e.g., ammonium sulphate, acetone, polyethylene glycol)
State 5 desirable properties in competing solutes
- Cheap and pure
- Not too viscous or dense
- Very soluble in water
- Relatively NON-DENATURING to proteins in general
- Easy to remove from the proteins afterwards
Briefly describe the principle of size exclusion chromatography
A protein sample is passed through a column containing carbohydrate polymer beads. Smaller proteins enter aqueous spaces within the beads, but larger ones cannot enter the beads and pass straight through the column more quickly
Briefly describe how ion exchange chromatography works
A protein sample is passed through a column containing charged beads - proteins with opposite charges to the beads bind to them, while proteins with opposite charge flow straight through. Bound proteins are then eluted by adding increasing concentrations of NaCl
Describe the principle of affinity chromatography
An immobilised molecule with affinity for the protein of interest is used to “trap” the desired protein in the column; other proteins flow straight through. Then, elution by adding competitor or changing conditions
Name (and explain in few words) the 5 types of affinity chromatography
- Immunoaffinity Chromatography (ANTIBODY against POI)
- Immobilised Ligand Affinity Chromatography (substrate analogue or inhibitor -> e.g., Heparin + Heparin-Binding Proteins)
- Lectin-Based Affinity Chromatography (lectins bind GLYCOSYLATED proteins)
- Immobilised Metal Affinity Chromatography IMAC (METAL IONS bind engineered recombinant proteins containing poly-His tag at N or C terminus; Fe3+/Al3+/Ga3+ can bind phosphorylated proteins)
- GST-Fusion Affinity Chromatography (GST = Glutathione-S Transferase, binds to immobilised GSH)
Describe the structure and key properties of the SDS molecule
Sodium dodecyl sulphate (SDS) is a detergent molecule, with dual ionic and hydrophobic character; it forms micelles in aqueous solutions and (at neutral pH) forms negatively charged, uniform shaped complexes with proteins in denatured form
State the three equations needed to calculate the values found in a protein purification table
Specific activity = total activity / total protein mass
Yield = total activity / ORIGINAL total activity
Purification Level = how many times the specific activity has increased by, compared to its original value
Describe (briefly) the principle of isoelectrofocussing
Proteins migrate towards either positive or negative charge (depending on their own charge) until they are at their isoelectric point and their own charge becomes 0
How does 2D Gel Electrophoresis work?
Proteins are separated horizontally by isoelectrofocussing, then vertically by Molecular Weight, effectively separating ALL proteins in the sample
What is proteomics?
The study and mapping of how protein expression differs between different cells (e.g., different cell types, treated/untreated cells, etc.)
Distinguish between monoclonal and polyclonal antibodies, and which is more useful in protein identification?
Monoclonal = uniform population of antibodies which all recognise the same epitope on an antigen
Polyclonal = mixed population of antibodies which recognise different epitopes on an antigen
[Monoclonal populations more useful in ID/separation]
Briefly describe the principle of Western blotting
After SDS-PAGE, proteins transferred to polymer sheet; sheet is then exposed to radiolabelled/enzyme labelled/luminescent, specific antibody; overlay photographic film, expose + develop; end up with Autoradiogram with a single band detected by the antibody
Describe a common use of immunofluorescence for identification
Cell Visualisation: actin filaments visualised with RFP-labelled antibody; GFP for MTs and DAPI for DNA in Nucleus
What is immunogold labelling used for?
Labelling proteins found in low densities, e.g., cell surface antigens
Which method is used for broad separation on the basis of size?
Dialysis
How does Immobilised Metal Affinity Chromatography (IMAC) work?
Metal ions bind to poly-His tag at the N- or C-terminus of modified proteins
How can metal 3+ ions (e.g., Al3+, Fe3+, Ga3+) specifically be used to bind proteins?
They selectively bind PHOSPHOproteins
