Electrophoresis Flashcards
What are the safety concerns when using gel electrophoresis?
Chemical toxicity
- Gel: Acrylamide monomer (neurotoxic, absorbed through the skin and destroys nerve endings. The polymer is safe, but to get that you must use the monomer)
- Buffer: Barbitone (barbiturate drug, so will fail police drugs tests. Also if spilt on you can act as a sedative)
- Stain: Ethidium Bromide (carcinogenic promoter, acts with a delayed effect)
Voltage and current
- Dry working conditions (if you’re stood in water the current will pass through you)
- Electrical cut offs on tanks
- Power supply recessed outputs
- sleeved plugs
What is electrophoresis?
Movement of charged molecules in solution from the application of an electric current.
Movement dependent on molecular size, shape and charge.
Speed molecule moves is directly proportional to the applied voltage.
Too much resistance = too much power = too much heat - can lead to gel setting on fire!
Mostly interested in the movement of proteins
- Amphoteric: charge depends on amino acid composition and pH. Charge is also changed by the side chain carboxyl and amino groups.
When analysing nucleic acids, which are negatively charged (due to acidic phosphate side groups acidic) they will always run towards positive side of gel (anode).
Barbital/Barbitone buffer is consistent at pH 8.6. At ph 8.6, normal serum will give 6 regions defined; (anode) albumin, alpha 1, alpha 2, beta 1, beta 2 and gamma (cathode).
How do you interpret the banding patterns from anode to cathode?
Thickness of band is proportional to concentration of protein present.
Albumin (always the heaviest band)
Alpha-1 zone:
a-1 antitrypsin, a-1 acid glycoprotein, a-1 antichymotrypsin
Alpha-2 zone:
Haptoglobin, caeruloplasmin, GC Globulin, a-2 macroglobulin, a-lipoprotein
Beta-1 zone:
Transferrin, haemopexin
Beta-2 zone:
B-lipoprotein, C3 complement
Diffuse gamma zone:
Immunoglobulins IgG, IgA, IgE, IgM and IgD
What are the advantages of using agarose in electrophoresis?
Serum protein electrophoresis is run on agarose as it gives good separation (has low endosmosis (way fluid runs through a gel - high gives ripples and things run off gel). Agarose also give low background and is adaptable to automated systems.
Although you then have to stain this gel so that you can see things, or the machine can.
What is a SEP scan?
Density metric scan on electrophoresis gel.
Measure total protein and work out AUC for each peak - tells us the concentration of proteins in each region, but not the specific protein.
Can quantitate monoclonal population with this method.
What does serum immunofixation involve?
Once we have identified a monoclone we need to work out what type of immunoglobulin is affected - do this through serum immunofixation. First lane is what you see in electrophoresis, the second is one that has been stained for IgG, then A and M and finally kappa and lambda.
What is capillary zone electrophoresis?
Gives density metric scan results, not using a gel anymore - all automated.
In contrast to serum electrophoresis, CZE detects the gamma region first due to detection flow direction.
CZE delineates an extra band between albumin and the alpha1 fraction, identified as triglyceride. The triglyceride band can be eliminated by a software correction.
Anomalies, such as a small IgG kappa monoclone, are more obvious in the CZE separation
What does a monoclonal gammopathy indicate?
Discrete band in the gamma region (more obvious in a CZE) is suggestive of (requires immunofixing and recognition of clinical picture and biopsy to look at B cells associated with a particular clone):
- Myeloma
- MGUS
- Waldenstroms macroglobulinaemia
- Lymphoma
- Infection
Myeloma: >10% plasma cells in bone marrow biopsy slide, Calcium increased (as osteoclast’s activity is increased = higher bone breakdown, leads to lyric regions and thin bones leading to pathological fractures), Renal function decreased (can get myeloma kidney), Anaemia, Bone lesions, Monoclonal protein/ Free light chain seen in serum electrophoresis.
Patient will have either:
- Smouldering myeloma - greater than 10% cells, but not all the clinical features YET.
- CRAM - all the above symptoms and greater than 10% cells = definite myeloma
- MGUS - myoclone of uncertain symptoms - less than 10% cells, but likely to turn into myeloma in the future eg 10 years)
What is the expected protein pattern in electrophoresis seen for Acute Phase Response (APR)?
↓Albumin band
↑Alpha-1
↑Alpha-2
Normal/ ↓Beta-1
Normal/ ↑Beta-2
Normal/ ↑Gamma
What are features of a chronic inflammatory pattern?
Increased:
A1AT, A2M, CPL, Fibrinogen, HPT, C3, A1AG, CRP, immunoglobulins
Decreased negative inflammatory proteins:
Albumin, Alpha-lipoprotein, Pre-albumin, Transferrin
Gives similar pattern to APR, but with more noticeable decrease in albumin and increased gamma region.
What are the features of a nephrotic pattern?
Increased:
A2M (alpha-2 microglobulin) - very large, so requires huge kidney damage to allow it to leak through
Decreased:
Everything else
Increased Alpha-2, all other bands decreased compared with normal.
What are the features of cirrhosis?
Increased - liver produced proteins:
A2M, CPL, Fibrinogen, Immunoglobulins - particularly IgA
Decreased:
Albumin, HPT, C3, TRF
Reduced Albumin, Alpha-1, Alpha-2, Beta-1, Beta-2 bands with increased Gamma region - IgA runs very close to beta-2, can cause gamma-beta bridging, where you don’t see a clear gap.
What is immunofixation electrophoresis?
Stain each immunoglobulin in the gamma region using amino black stain.
Can more easily identify IgA lambda monoclonal gammopathies.
What is lipoprotein electrophoresis, and what is the difference between normal and abnormal?
Lipoproteins composed of protein and lipid.
Transport triglycerides and cholesterol.
Increased levels associated with cardiovascular disease.
Serum or EDTA plasma (not heparin plasma).
SEP
Stain
Read/Scan
In a normal lipoprotein EP there will be normal cholesterol and triglyceride values, a beta lipoprotein major band, pre Beta faint or absent, Alpha band less intense than Beta. Means more HDL than LDL.
In abnormal lipoprotein EP there is often an elevated cholesterol or triglyceride value. Split abnormality into Primary or secondary disease
5 classes primary lipoproteinaemia (Fredrickson classification).
- Type 1: Chylomicrons present, decreased alpha and beta.
- Type 2: Increased beta with normal (2a) or increased pre beta (2b). (cardiovascular disease)
- Type 3: Broad beta band. (cardiovascular disease)
- Type 4: Increased pre beta. (cardiovascular disease)
- Type 5: Chylomicrons present, pre beta increased.
How and why are ALP isoenzymes measured?
ALP present in many tissues – bone, liver, intestine, placenta. (hydrolyses PO4 esters)
Serum ALP is a composite of iso enzymes - Don’t know where it has come from.
ALP increased- cholestasis (obstructive jaundice, malignancy), osteoblast hyperactivity and bone remodelling (Pagets, fracture, malignancy). Also Hodgkins, coeliac, UC, regional arteritis etc.
Measure using a serum or heparin plasma sample (6not EDTA plasma) SEP Stain Read/Scan Need total serum ALP value
