Electrophoresis Flashcards
What is electrophoresis?
The motion of charged particles in a colloid under the influence of an applied electrical field.
State factors influencing migration
Size (radius), shape, charge, viscosity, electrocal field strength, temperature, gel effects (e.g. endosmotic flow)
State components
Power supply (electrode and anode), Sample applicator, buffer, temp control, detection system, gel/matrix
How does high voltages affect the migration rate?
Higher voltage=faster migration
- BUT high voltages also mean increased current and resistance
- Resistance generates heat, which has the potential to denature proteins
How is resistance generated by voltages managed?
Cooling system
- Could be a cold room or using a peltier device (heat absorbed at junction between materials)
Peltier for capillary
ADDITIONAL, what is a peltier device?
Ceramic plates seperated by layers of bismuth and telluride semi-conducters
- Current passed through junction between semi-conductors
- This generates a cool side of the device which absorbs heat
Function of buffer?
Carries current, controls pH and molecular charge
How can the resolution of electrophoresis be enhanced? By modulating the buffer only
Greater ionic strenght the better resolution
- However, this increases the number of charges flowing increasing heat
Stacking at buffer boundary improves resolution
What type of gel matrix is most suitable for DNA and proteins?
DNA (Long chain) - agarose
Protein and short DNA reads- polyacrylamide
How does the concentration of agarose affect pore size?
Higher % agarose result in smaller pores
Typically use 0.5-2% agarose
Describe the structure of polyacrylamide.
Chemically crosslinked chains of acrylamide and bisacrylamide
- Total % and ratio of acryl:bisacryl determines pore size
- 5-15% typically used for gels
What is the requirement of sample to be run in electrophoresis?
It needs to be charged
- Can be altered using pH or adding of SDS
DNA is ideal, since it has a native negative charge - may need to amplify, sonicate, digest and label
Why is serum a better sample type than plasma?
Fibrinogen will interfere and produce an unwanted peak in plasma
How does urine need to be prepped?
Pre-concentrated
- Using a vivaspin device which has a microfilter that retains proteins and removes excess water
- Also use morning sample - more concentrated naturally
- Creatinine is a marker for concentration
How is detection of electrophoresis carreid out?
- UV/visible spectrophotometry - e.g. peptide bond absorbs in UV range 220nm
- Dyes - coomassie brilliant blue
- Exploit molecular properties e.g. enzyme and dye substrate, use lectins for glycosylated molecules
- Fluoresence and chemiluminescence is the most sensitive method
What is endosmotic flow?
This occurs in all systems of electrophoresis, but is most prominent in capillary electrophoresis.
- Glass capillaries contain silica groups carrying small negative charge when voltage is applied. This attracts positive ions in buffer to 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
- Endosmotic flow dominates migration because surface area is big
Basically - Ions move the opposite way we are expecting them to
Use albumin to explain endosmotic flow?
Albumin based purely on charge, should migrate towards the cathode (+), but it actually migrates towards the anode(-).
Because positive ions of the buffer coat the cathode producing a positive charge
Match the following troubleshootings of electrophoresis.
A. Discontinuities in bands
B. Unequal migration on gel
C. Areas of gel faded/washed out
- Broken or dirty sample collecter, bubbles in gel
- Gel too wet, sample not concentrated or overconentrated
- Faulty electrode, uneven wetting on gel
A1
B3
C2
Draw and label the graph produced from a serum electrophoresis diagram.
6 different peaks, first one is much greater than the others.
- In order: albumin, alpha-1, alpha-2, beta-1, beta-2 and gamma
Match the peaks with assocaited proteins:
A. Alpha-1 B. Alpha-2 C. Beta-1 D. Beta-2 E. Gamma
- Hemopexin, transferrin
- Haptoglobin, Alpha-2-macroglobulin
- Immunoglobulin
- Alpha-1 acid glycoprotein, alpha-1 antitrypsin
- C3 complement
A4 B2 C1 D5 E3
How is the serum albumin electrophoresis peaks affected during an inflammatory response?
Albumin decreases, but all other increase
Increase will appear as thicker bands
How is the serum albumin electrophoresis peaks affected in liver disease?
Albumin decreases
Increase in beta 2
Note: gamma may see fused beta/gamma (bridging)
How is the serum albumin electrophoresis peaks affected in nephrotic syndrome?
Decrease in albumin and gamma
Increase in alpha-2, because its being retained in kidneys
How is the serum albumin electrophoresis peaks affected in haemolysis?
Increase in alpha-2 and beta-1
How is the serum albumin electrophoresis peaks affected in hypogamma-globulinaemia?
Decrease in gamma
How is the serum albumin electrophoresis peaks affected in Myeloma?
Decrease in albumin
Increase in gamma (sharp peak, similar to albumin)
- Because you have the production of one singular type of Igs which all migrate to the same place
What is multiple myeloma?
Bone marrow cancer due to proliferation of plasma cells, which produces Igs in excess.
Major features:
- Calcium, hypercalcaemia due to lysis of bone
- Renal impairment - due to immunoglobulins clogging kidney
- Anaemia, as plasma cell dominate bone marrow
- Bone, lytic lesions as cells stimulated to break down bone
State some diagnostic features of myeloma.
- Bone marrow biopsy is necessary (plasma cell > 10%)
- Lytic lesions on CT scan
- Haemoglobulin level (hyper), calcium (hyper), urea (raised) and creatinine (raised)
- Serum and urine electrophoresis
What are some related conditions of myeloma?
Monoclonal gammopathy of undetermined significance (MGUS)
Smouldering myeloma
Plasmacytoma
What is Monoclonal gammopathy of undetermined significance (MGUS)?
Monoclonal gammopathy of undetermined significance (MGUS) - small paraprotein but plasma cell <10% and no clinical features. 1% progress to myeloma
What is smouldering myeloma?
Paraprotein and plasma cell proliferation but no clinical features
What is plasma cytoma?
Plasma cell tumour outisde bone marrow. No paraprotein, normal bone marrow and no end organ damage
Match the following
A. Polyclonal
B. Oligoclonal
C.Monoclonal
- Lots of plasma cells making different types of Igs
- Cancerous cells originate from a single abnormal cell and make the same Ig
- Certain types of plasma cells proliferate because some types of Igs are better than others
A1
B3
C2
Describe the polyclonal pattern of albumin electrophoresis
Gamma peak has increased and has a more defined peak, which interferes with reading the beta-2 peak slightly. Whilst albumin is slightly lower
- Gamma is broad because many different types of Igs are produced
Describe myeloma pattern of albumin electrophoresis
Albumin peak has decreased, whilst gamma has increased massively to surpass albumin.
- Gamma has an extremely define peak because only one type of Igs is made
Following screening of myeloma using the serum albumin electrophoresis, what can be done?
Skeletal servey to look for skeletal lesions, but generally urine electrophoresis is requested.
- Used to look for bence jones portein
- When they are present it is usually poorly prognostic - Gel is the most sensitive method of detection
- Single abnormal band - myeloma
Following detection of Bence Jones protein, what is appropriate course?
Immunofixation to confirm the protein is there
- Gel based method using six parallel lanes
- Antisera precipitate any antibodies in lane 2-6
- All other proteins are removed
- Only Igs of the type corresponding to antiserum added are trapped on gel and show up when stained
Match the following
A. Lane 1
B. Lan2-4
C. Lane 5-6
- Normal electrophoresis + protein fixative
- Antibodies to Kappa and Lambda light chains
- Antibodies to IgG, IgA and IgM added
A1
B3
C2
Advantages of immunofixation
Removes background staining
Identifies type of Ig present
Removes interfering substances (e.g. fibrinogen in plasma samples)
Function of antisera in immunofixation.
Precipitate any antibodies present in lanes 2-6
What is immunosubraction?
Similar to immunofixation but opposite way around. Alternative to identify Igs
What is immunofixation?
Running the serum containing the Igs on a gel. Application of electrical field will seperate the Igs according to electrophoretic mobility
- Make them migrate under the effect of an electric field - Migration is based on mass and charge
- Antisera is placed individually to each migration lane
- Presence results in appearance of a narrow band after staining complex precipitates
Match for Immunosubtraction
A. Capillary 1
B. Capilalry 2-4
C. Capillary 5-6
- Sample + FK/FL antisera
- Sample + Ig G, A, M antisera
- Normal sample
A3
B2
C1
How is antisera utilised in immunosubtraction compared to immunofixation?
Retain band vs remove it - Hence the name
In immunosubtraction the antibodies react with their corresponding antisera and are substracted from trace - because it migrates differently to the sample
- Disappearance of the abnormality in the antiserum treated patter indicates the presence of a monoclonal protein
In immunofixation the antisera is used to detect the antibody
How is the immunosubtraction analysed?
Trace with another colour on top of the myeloma trace (5 peak). If the new trace does not show peak at gamma then the corresponding Igs is present
What is likely to cause unusual bands or peaks?
- Haemolysis - Haemoglobin migrates in beta
- Fibrinogen - extra band seen in whole blood
- Extra peaks in albumin zone - antibiotics bisalbuminaemia
- Sample ageing/denaturation
- Capillary ageing or contamination
Using an example how can isoenzyme analysis be achieved using electrophoresis?
e. g. ALP
- Exist in bone, liver, placenta, intestine
- Thus raised levels are hard to interpret
- Bone ALP is heavily sialylated/glycosylated, liver is not
- Seperation using electrophoresis is acheived by different levels of glycosylation
- Lectin binds sialic acid and affects migration of bone ALP (dense bands at bottom of gel)
Describe isoelectric focussing.
- Exploits fact that particle are only influenced by an electric field if charged
- pH gradient created by charged molecules named ampholytes
- As pH decreases, amino acid side chains bind H+ in gel
- Acidic chemical groups within the protein loose their charge and basic group become positively charged
The ratio between basic and acidic groups will determine where on the electrophoresis they end up. The position has a corresponding pH. This is the isoelectric point (Pi).
Small differences in amino acid sequence can change Pi of protein - Isoelectric focussing will allow seperation of these
Function of alpha-1-antitrypsin in inflammation
Alpha-1-antitrypsin is produced by the liver and act to inhibit proteases from breaking down tissue, e.g. elastin in lungs.
- Alpaha-1-antitrypsin defiencency describes the condition where you lack the enzyme to inhibit proteases
State some other clinical uses of electrophoresis and isoelectic point focussing
- HbA1c: glycated haemoglobin for monitoring diabetes - serparate from other Hb fractions
- Transferrin Variants - detect glycosylation disorders of alcohol abuse (affects glycosylation)
- Identification of unknown body fluids in CSF leaks
- Immunology - original diagnosis of agammaglobulinaemia
