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
How can multiple myeloma be clinically diagnosed?
* 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
What are conditions related to multiple myeloma?
* **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
How can multiple myeloma be diagnosed by urine electrophoresis?
* **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
How is immunofixation produced?
* 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
How does immunofixation appear in serum and urine samples?
* **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
What is immunosubtraction?
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
What are causes of unusual bands or peaks?
* **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
What is Isoelectric focusing?
* 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
How is alpha 1 anti trypsin produced?
* 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
How is Alpha 1 antritrypsin deficiency treated?
Over 90 variants known
* **Treatment:** Recombinant AAT (milk, rice)
* **Cure:** Transplant / gene therapy ???
35
What are some other clinical uses of gel electrophoresis?
* **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
