Electrophoresis Flashcards
1
Q
What is Electrophoresis?
A
The motion of charged particles in a colloid under the influence of an applied electric field
2
Q
What are factors that influence migration?
A
- Size (radius)
- Shape
- Charge
- Viscosity of the colloid
- Electrical
- Field Strength (potential diff./ voltage)
- Temperature
- Gel Effects (e.g. endosmotic flow)
3
Q
What are components of an Electrophoresis system?
A
- Power Supply
- Temperature Control
- Buffer
- Detection system
- Gel/Matrix
- Sample Applicator
4
Q
How does the power supply influence the electrophoresis system?
A
- Higher voltages lead to faster migration and better turn around times
- Capillary electrophoresis uses huge voltages typically kV (i.e. 10-100 x mains)
- May require non-standard power supply
- High voltages can lead to current resistance
- Resistance generates HEAT which can denature molecules and influence migration
5
Q
What are cooling systems used in Electrophoresis systems?
A
Needs strict temperature control
- Traditional gels = stirring block + cold room
- Capillary electrophoresis uses a ‘Peltier device’ (heat absorbed at junctions between materials)
6
Q
What is the purpose of buffer in electrophoresis?
A
- Carries current, controls pH and molecular charge
- Can add other molecules to influence migration e.g. SDS (denatures), ampholytes (pH gradient)
- ‘Stacking’ at buffer boundary improves resolution.
7
Q
What are types of gel or support matrices?
A
Several types available depending on type and size of molecule to be separated, resolution required Starch / Cellulose - brittle, need pre-soak • Agarose typically used for DNA • Polyacrylamide popular for proteins
8
Q
How can gel or support matrices be influenced?
A
- Can add molecules (e.g enzyme substate) to allow detection or influence migration
9
Q
What are features of Agarose gels?
A
- Complex polysaccharide derived from seaweed.
- Higher % agarose results in smaller pores (0.5-2%)
10
Q
What are features of Polyacrylamide?
A
Chemically crosslinked chains of acrylamide and bisacrylamide. • Total % and ratio of acryl : bisacryl determines pore size
11
Q
What are benefits and drawbacks of Agarose gels?
A
Benefits
- Cheap
- Non-Toxic
- Can prepare in-house
- Sets rapidly
- Ideal for longer DNA 50-20000bp
Drawbacks
- Non-uniform pore size
- Gels weak at low % agarose
- Gel brittle at higher % agarose
- No CE-marking
- May not set evenly
12
Q
What are benefits and drawbacks of Polyacrylamide gels?
A
Benefits
- Uniform pore size, Reproducible results
- High resolving power
- Stronger, thinner gels dissipate heat better
- Chemically inert
- Ideal for short DNA (5-500 bp) and proteins
Drawbacks
- Acrylamide is neurotoxic
- Longer time to wait until set
- More expensive
- Need to buy gels in from a company
13
Q
What sample types can be used for electrophoresis?
A
- Need charged molecules (alter pH or add SDS if not)
- DNA ideal due to native negative charge – may need to amplify, sonicate, digest, label etc.
- Proteins can be run natively (large complexes) or with SDS (adds -ve charge, ensures migration due to size only)
- For serum, avoid haemolysis, fibrinogen, contrast media and other interfering substances
- Urine / CSF samples may need to be pre-concentrated
14
Q
How is Urine assessed?
A
- Ideally an early morning urine
- Assess concentration using urine creatinine
- Vivaspin® urine concentrators for dilute urine samples e.g. elderly patients on diuretics, Centrifuge sample
- Microfilter retains proteins and removes excess water
15
Q
How is the sample applied to the Gel electrophoresis?
A
- Usually around 2-50μL sample
- Can be done manually with a loading dye
Most automated systems use either:
- (1) Electrokinetic Application where a small voltage applied to drive sample into buffer,
- (2) Hydrodynamic application where positive pressure applied ‘pushes’ sample into buffer
16
Q
What are the section systems used in Gel Electrophosiss?
A
- UV/visible spectrophotometry e.g. peptide bond absorbs in the UV range (220nm)
- Dyes e.g. Coomassie Brilliant Blue, Silver Nitrate + ‘densitometry’
- Exploit molecular properties e.g enzyme + dye substrate, use lectins for glycosylated molecules
- UV/visible spectrophotometry e.g. peptide bond absorbs in the UV range (220nm)
- X-ray or UV Imaging e.g. ethidium bromide for DNA
17
Q
What is Endosmotic Flow?
A
- Happens to small extent in all systems but especially prominent in capillary electrophoresis
- Glass capillarys contain silica groups which carry a small negative charge when voltage is applied
- This attracts positive ions in the buffer which align on capillary walls
- Rearrangement of ions in buffer to align with walls causes a ‘tide’ to flow in the opposite direction to electromotive force apillary surface area is big, so endosmotic flow dominates migration in capillary systems
- Molecules are carried in the opposite direction to what is expected based on size and charge
- Serum samples run on gel show albumin band by the anode and smaller proteins closer to the origin
- Serum samples run through capillary systems show smaller proteins on the cathodal side of the origin
18
Q
What are possible issues that can occur in Electrophoresis and their causes?
A
- Discontinuities in bands
- Unequal migration on Gel
- Areas of gel faded/washed out unuausual bands and peaks seen
19
Q
What are causes of Discontinuities in bands?
A
- Broken or dirty sample applicator
- Bubbles in gel
- Application marks
20
Q
What are causes of Unequal migration on Gel?
A
- Faulty electrode
- Discontinuities in buffer or gel
- Uneven wetting of gel
21
Q
What are causes for Areas of gel faded/washed out?
A
- Gel too wet
- Sample not concentrated enough (Urine/CSF)
- Sample over-concentrated (leads to pale region in the middle of very dark band)
22
Q
What are monoclonal gammopathies?
A
- Group of disorders characterised by the proliferation of a single clone of plasma cell that accumulate within the bone marrow.
- Results in the production and appearance of a monoclonal protein (paraprotein)
- Excessive proliferation of a single plasma cell clone can suppress healthy plasma cell production leading to reduced polyclonal immunoglobulin levels
- Immunoparesis (secondary immunodeficiency)
23
Q
What is a multiple myeloma?
A
- Bone marrow cancer due to proliferation of plasma cells
- Plasma cells make excessive amounts of immunoglobulin
- Accounts for 2% of cancers in the UK - 5,700 new cases/year (>65s)
24
Q
What are symptoms of Multiple myeloma?
A
- Calcium: Hypercalcaemia due to lysis of bone
- Renal impairement: Due to immunoglobulins ‘clogging’ kidney
- Anaemia: As plasma cell dominate bone marrow
- Bone: Lytic lesions as cells stimulated to break down bone
25
Q
How can multiple myeloma be clinically diagnosed?
A
- Bone marrow biopsy (plasma cells >10%) 2)
- Lytic lesions on Ct scan or X-ray
- Haemoglobin level, calcium, urea, creatinine
- Serum and urine electrophoresis
26
Q
What are conditions related to multiple myeloma?
A
- Monoclonal Gammopathy of Undetermined Significance (MGUS): Small paraprotein but plasma cells <10% and no clinical features (1% progression to myeloma per year)
- Smouldering Myeloma: Paraprotein and plasma cell proliferation but no clinical features
- Plasmacytoma: Plasma cell tumour outside bone marrow. No paraprotein, normal bone marrow and no end organ damage
27
Q
How can multiple myeloma be diagnosed by urine electrophoresis?
A
- When healthy plasma cells make immunoglobulin, they secrete intact Immunoglobulin (two heavy and two light chains), but also an excess of light chains.
- Normally these excess free light chains are reabsorbed in the proximal tubules of the kidney and metabolised (Not detected in urine of healthy individuals as all reabsorbe)
- However if myeloma plasma cells are producing large amounts of free light chains, this can overwhelm the kidneys (Free light chain excreted at detectable levels in the urine)
- Look for ‘Bence Jones Protein’ – immunoglobulins light chains in urine
28
Q
How is immunofixation produced?
A
- Patient serum run on gel in six parallel lanes (Lane 1: normal electrophoresis + protein fixative and IgG, IgA, IgM, Kappa and Lambda light chains
- Antisera precipitate any antibodies present in lanes 2-6
- All other proteins are removed by blotting and washing.
- Only immunoglobulins of the type corresponding to the antiserum added are trapped on gel and show up when stained
- Removes background staining (more sensitive)
- Identifies type of Ig present
- Removes interfering substances (fibrinogen, contrast dye)
29
Q
How does immunofixation appear in serum and urine samples?
A
- Serum: Both show IgGλ paraprotein plus monoclonal λ free light chain
- Urine: Presence of large intact monoclonal immunoglobulin in urine (in addition to smaller free light chain) demonstrates kidney damage
30
Q
What is immunosubtraction?
A
Similar principal (but opposite way round). Electrophoresis run through 6 capillarys
- Capillary 1 = normal sample
- Capillarys 2-4 = sample + Ig G, A, M antisera • Capillarys 5-6 = sample + FK / FL antisera
- Antibodies react with their corresponding antisera and are ‘subtracted’ from trace
- Disappearance of the abnormality in the antiserum-treated pattern indicates the presence of a monoclonal protein.
31
Q
What are causes of unusual bands or peaks?
A
- Haemolysisis: Haemoglobin migrates in beta
- Fibrinogen: Extra band seen in whole blood
- Extra peaks in albumin zone: Antiobiotics bisalbuminaemia, contrast media, bile salts
- Sample ageing/denaturation
- Capillary ageing or contamination
All of the above will not show a convincing band on immunofixation/subtraction. If in doubt, fix!
32
Q
What is Isoelectric focusing?
A
- Exploits the fact that particles are only influenced by an electrical field if charged
- Gel used has a pH gradient created by charged molecules named ‘ampholytes’
- Most proteins have a net positive or negative charge, depending on amino acid side sequence
- As pH decreases, side chains bind H+ in gel.
- Acidic chemical groups within the protein loose their charge and basic groups become positively charge At a certain pH, the number of positive and negative charges will balance and the protein has no net charge
- This is known as the ‘isoelectic point’ (Pi)
- When the protein reaches it’s isoelectric point, it carries no net charge and is no longer influenced by the electrical field i.e. it stops migrating
- Small differences in amino acid sequence can change the isoelectric point of a protein
- Isoelectric focussing will allow separation of these
33
Q
How is alpha 1 anti trypsin produced?
A
- During inflammation, neutrophils release powerful proteases to cleave toxins etc.
- Unfortunately, these can also react with human proteins, such as elastin, in the lungs.
- Alpha 1 antitrypsin is synthesised by the liver and released into plasma during inflammation
- Inhibits elastase and protects tissue from damage
- Mutations can decrease Alpha-1-AT level or alter structure
- Exacerbated in smokers due to increased lung inflammation and neutrophil recruitment
34
Q
How is Alpha 1 antritrypsin deficiency treated?
A
Over 90 variants known
- Treatment: Recombinant AAT (milk, rice)
- Cure: Transplant / gene therapy ???
35
Q
What are some other clinical uses of gel electrophoresis?
A
- HbA1c: Glycated haemoglobin for monitoring diabetes - separate from other Hb fractions
- Transferrin Variants: Detect glycosylation disorders or alcohol abuse (affects gylcosylation)
- Identification of unknown body fluids in ?CSF leak (desialated β2 transferrin a.k.a. ‘Tau protein’ is unique to CSF and distinguished from nasal fluid)
- Detection of oligoclonal bands in the CSF and serum of patients with Multiple Sclerosis (isoelectric focussing)
- Detection of other isoenzymes (CK, amylase)
- Lipids: electrophoresis of serum samples with a specific stain for lipids (rare) Unusual patterns associated with metabolic disorders affecting lipid metabolism
