electrophoresis

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Notes on electrophoresis.. By Dr. Ashish V. Jawarkar Contact: [email protected] Facebook: facebook.com/pathologybasics

ELECTROPHORESIS

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OVERVIEW

1. History 2. Zonal electrophoresis

Principle Method

3. Buffer 4. Support media 5. Ancillary techniques before performing electrophoresis

Precipitation Column separation

- Gel filteration - Ion exchange chromatography (anion exchange, cation exchange) - Hydrophobic chromatography - Affinity chromatography - Capillary electrophoresis

6. Summary of workflow 7. Applications

a. Serum protein electrophoresis b. CSF electrophoresis c. Urine electrophoresis d. Hemoglobin electrophoresis

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* HISTORY

1. The technique of electrophoresis was introduced by Tiselius. 2. His technique is known as MOVING BOUNDARY ELECTROPHORESIS / FRONTAL

ELECTROPHORESIS 3. He separated proteins dissolved in electrolyte solution by application of electric current

through U shaped quartz tube that held the solution. 4. At ph 7.6, the solution got separated into four distinct protein fractions, albumin, α, β

and γ. These separated proteins were identified by checking the change in refractive index across the solution.

5. As there was no solid support to the medium, these separated proteins did not form distinct zones.

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* ZONAL ELECTROPHORESIS 1. In Teselius’ technique, the proteins cound not form distinct bands 2. This problem was solved by introducing an anticonvection solid support medium – like

filter paper. 3. Now on applying electric current, the proteins separated into discrete bands at ph 8.6 4. The α band got further split into α1 and α2. 5. Other solid media that are frequently used nowadays include cellulose acetate

membrane, agarose gel, starch gel and polyacrideamide gel.

Anode Cathode

Principle: 1. The proteins to be separated are applied at anode. 2. The support medium has negative charge – the electromotive force (determined by

charge of proteins and pH) tends to move the support medium towards anode (positive pole) – but support medium cannot move.

3. The buffer ions have positive charge – they tend to move towards cathode. 4. This net result is flow of buffer towards cathode – this force called electro osmotic force

(endosmosis). 5. The buffer ions carry with them water molecules and protein molecules. Proteins here

move not only on the basis of their charge, but also determined by their molecular weight.

6. This explains the movement of slightly negatively charged γ globulins towards the cathode.

7. The actual distance traveled by the protein is dependent on these two forces described above.

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Method: 1. The sample to be separated is soaked into a gel. 2. Each end of the gel is then inserted into separate buffer chambers in which electrodes

are mounted. 3. An electric current is then passed through the gel for a period of, about 30 minutes

usually. 4. After this the gel is treated with a mild fixative such as acetic acid. 5. This fixative precipitates the proteins at the positions on the gel to which they have

migrated to. 6. They are then stained and the gel is dried. 7. The protein patterns can now be visually inspected for qualitiative identification of

abnormal proteins. 8. To find out the quantity of each type of protein present, densitometric scanners are

used, they generate tracings and quantitate relative percentages of proteins in each fraction.

9. These percentages when multiplied by total proteins can give an idea about the concentration of protein in each fraction.

Eg. If total protein is 9 gm/dl and albumin fraction obtained is 50% - = 4.5 gm/dl (9 x 50/100) is the albumin concentration 10. On varying the buffer media / solid media – different applications of electrophoresis can

be developed.

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* Varying buffer concentration and its application 1. If endosmosis is set at a high, this maximizes cathodal movement. 2. Immunoglobulins can be separated optimally and this helps in detecting oligoclonal

bands in CSF

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* Varying solid media and its applications PAGE – Polyacrylamide Gel Electrophoresis

1. Polyacrylamide is an inert support medium 2. It is useful for standard separation of native proteins based on charge

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SDS-PAGE – Sodium dodecyl sulphate Polyacrylamide gel electrophoresis

1. SDS denatures proteins according to their molecular weight 2. SDS-PAGE is very powerful for resolving proteins and separating them into subunits

according to their molecular weight 3. Very useful for research purposes

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TWO DIMENSIONAL ELECTROPHORESIS (2D ELECTROPHORESIS) 2. Standard separation of proteins is done in one direction 3. This is followed by SDS-PAGE in perpendicular direction 4. This results in hundreds of identifiable protein peaks

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ISOELECTRIC FOCUSSING 1. Gives superior resolution to closely migrating proteins 2. Here the gel used contains a gradient of pH established by using ampholytes 3. As each protein reaches the gel location where the pH equals its own, the net charge

becomes zero 4. It cannot move further 5. Thus proteins move here strictly on the basis of their pH

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* ANCILLARY TECHNIQUES PRECIPITATION 1. The sample to be tested usually contains a lot of undesired proteins. 2. These undesired proteins can be removed by precipitation so that a single minor protein

can be isolated by electrophoresis. 3. First the value of total proteins is found out 4. Then Sodium sulfate, sodium sulfite, ammonium sulfate or methanol is used to

precipitate globulins. 5. Protein estimation from the remaining solution after precipitation can give an idea

about the albumin concentration – electrophoresis can give an idea about the types of proteins apart from globulins.

6. globulin = total protein – albumin 7. estimation of albumin : globulin ratio is useful in various disorders Normal :

A:G ratio >1:5 (1-3)

Decreased A:G ratio Increased A:G ratio Decreased albumin Malnutrition Malabsorption Liver failure Proteinuria Accumulation of ascetic fluid Enteropathy Increased globulins Acute or chronic infections Myelomas

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COLUMN SEPARATION

(1) Gel filteration:

1. Media such as agarose beads/sephadex is used 2. They have beads in them with pores of various sizes 3. Largest proteins cannot pass through pores in the beads – they pass through spaces in

beads and are eluted first 4. Medium sized proteins pass through medium sized pores and appear second. 5. Small sized proteins pass through small sized pores and are eluted last.

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(2) Ion Exchange Chromatgraphy 1. This is of two types – Anion exchange and Cation exchange chromatography 2. This technique uses charged particles which are placed in the column. 3. When proteins having the opposite charge as the column pass through it, they are

retained for a longer time, neutral elute the second while same charged particles elute first.

ANION EXCHANGE CHROMATOGRAPHY 1. Media such as DEAE or QAE are used with have net positive charge. 2. Albumin, α and some β globulins which are anions stick to positively charged matrix. 3. γ globulins that have no charge are eluted faster. CATION EXCHANGE CHROMATOGRAPHY 1. Media such as carboxymethylcellulose are used which have a negative charge. 2. Positively charged cations stick to the matrix 3. Albumin is eluted first here

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(3) Hydrophobic chromatography 1. In this technique, support medium interacts with proteins according to their

hydrophobic nature. 2. Here ‘high salts’ and ‘low salts’ are used 3. high salts structure water around themselves and expose the hydrophobic ends of

proteins 4. these hydrophobic ends get attracted toward the medium and stick to it 5. according to hydrophobic nature of proteins, they stick or are eluted 6. ‘low salts’ are used to elute as they donot / expose less number of hydrophobic ends.

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(4) Affinity chromatography 1. This is based on specific binding between a protein of interest and another protein (can

be its antibody/antigen) bound to the solid medium. 2. Bound protein can then be eluted in its purified form. 3. examples

a. to separate FVII-vWF – column is incorporated with monoclonal antibody to vWF

b. Igs can be separated by using Staphylococcal protein A in gel matrix c. Attaching dihydroxyboronate to medium helps separate glycosylated

hemoglobin d. To separate digoxin – digibind is used

CroFab – crotalid snake venom

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(5) Capillary electrophoresis

1. This procedure is similar to HPLC in that a column is employed where separation of molecules is done on the basis of size/hydrophobicity or strereospecificity.

2. Applicable to large molecules such as DNA.

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* SUMMARY OF WORK FLOW

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* APPLICATIONS (1) SERUM PROTEIN ELECTROPHORESIS Following patterns are observed in normal conditions and various disorders on electrophoresis. Normal:

Cirrhosis of liver:

When liver function is sufficiently diminished, protein synthesizing capacity is compromised and concentrations of albumin and proteins in the alpha and beta bands are decreased. An additional common finding is beta-gamma bridging due to increased IgA.

Beta-gamma bridging

Alb α1 α2 β γ

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The Nephrotic Syndrome -- 1. Renal disease involving the glomeruli is always associated with increased urinary protein

loss. When protein loss is greater than 3-4 g/day, the protein synthesizing capacity of the liver is exceeded and hypoproteinemia, accompanied by anasarca, develops to cause the nephrotic syndrome.

2. The massive urine protein loss is due to increased permeability of glomeruli to protein. The permeability increase may be minimal so that only albumin and other smaller molecular weight proteins are selectively filtered (selective nephrosis, as in Minimal Change Disease) or may be greater so that larger proteins are also filtered (nonselective nephrosis, as in membranous golmerulonephritis) as is the case in the example shown.

3. Alpha-2-macroglobulin is sufficiently large so that it is not filtered and increased synthesis (from the general hepatic protein synthesis) causes its accumulation.

4. Lipoproteins are also sufficiently large to accumulate and hyperlipidemia is a characteristic of the nephrotic syndrome, although lipoproteins are not stained with the protein stain used in visualizing proteins.

Normal

Abnormal

Alb α1 α2 β γ

Alpha-1-Antitrypsin Deficiency: A genetic defect causes a deficiency of alpha-1-antitrypsin. The antiprotease deficiency

results in a propensity to develop emphysema. Since alpha-1-antitrypsin is the major component of the alpha-1 band, deficiency is suggested by a reduced alpha-1 band. Deficiency is confirmed by specific immunochemical quantification.

Normal

Abnormal

Alb α1 α2 β γ

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Acute Inflammation

The alpha-1 and alpha-2 bands are increased during the inflammatory response from increased hepatic synthesis of acute phase reactant proteins.

Normal

Abnormal

Alb α1 α2 β γ Chronic Inflammation --

Immunoglobulin synthesis by antigen activated B lymphocytes transformed to plasma cells is demonstrated by the increased polyclonal gamma band. Normal

Abnormal

Alb α1 α2 β γ

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Immunoglobulin Deficiency --

Deficient immunoglobulin synthesis is revealed by a markedly diminished gamma band. Effected individuals are prone to recurrent infection Monoclonal Gammopathy --

1. An unusually sharp band in the gamma region strongly suggests the presence of a homogeneous immunoglobulin and, thus, the malignant proliferation of plasma cells from a single cell (multiple myeloma) in contrast to the broad, heterogeneous, or polyclonal, gamma band as exhibited above in chronic inflammation from immunoglobulin synthesis by many different clones of plasma cells.

2. Homogeneous immunoglobulins are also found in Waldenstrom's macroglobulinemia (where the sharp gamma band is always IgM). Specimens which exhibit a narrow gamma band are further examined by immunofixation electrophoresis as described below

Alb α1 α2 β γ

(2) CSF ELECTROPHORESIS

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1. The diagnosis of Multiple sclerosis is ultimately a clinical one based on neurologic history and physical examination.

2. Nevertheless, advanced laboratory results such as elevated IgG indices and presence of oligoclonal bands of IgG or Kappa /lambda light chains on CSF electrophoresis, as well as neuroimaging techniques have proven to be invaluable in diagnosis of MS.

Agarose gel electrophoresis

1. Concentrated CSF is used as sample 2. Shows discrete bands of IgG – the oligoclonal bands 3. Presence of two or more bands is necessary for diagnosis 4. False positive results are obtained in SSPE, CNS infections like neurosyphilis,

Crypotococcal meningitis, GBS, transverse myelitis, burkitt’s lymphoma etc. 5. Sensitivity is less than other methods of electrophoresis Staining Commassie brilliant blue/paragon violet/silver staining 1. silver staining is more sensitive then CBB 2. however these techniques must be simultaneously carried out on patients serum also

because polyclonal gammopathy can lead to diffusion of immunoglobulin into CSF and lead to false positive results. This is seen in case of liver diseases, SLE, RA and chronic granulomatous diseases.

ImmunoFixation Electrophoresis

1. Immunofixation electrophoresis (IFE) is used to demonstrate that a narrow gamma band is due to a homogeneous immunoglobulin.

2. In the illustration below, 6 replicates of the specimen are loaded on to separate lanes of an IFE gel.

3. Following electrophoresis, the protein in each lane is stained differently. 4. The first lane (SP) is stained for total protein. 5. The protein in each of the other lanes is "stained" with specific antisera for

immunoglobulin heavy and light chains, respectively, as illustrated in the figure. 6. The finding of the preponderance of only one light chain associated with a

predominantly staining heavy chain confirms the molecular homogeneity of the immunoglobulin and also provides identification.

7. IFE identifies the narrow band as monoclonal IgG, lambda. 8. Sometimes malignant plasma cells synthesize excess light chains and less frequently

only light chains are synthesized. Almost never is excess heavy chain or only heavy chain synthesized. Excess free lambda light chain is exhibited in the illustration.

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a. Lymphocytic pleocytosis and 2 plasma cells in the CSF cytospin preparation. b. 4b. Isoelectric focusing/immunofixation revealed CSF-restricted oligoclonal IgG

bands as well as identical paraprotein bands in CSF and serum. Anode is on the left.

c. 4c. Serum immunofixation electrophoresis revealed the presence of IgGκ monoclonal band (arrow). Anode is at the top. ELP, electrophoresis (general protein stain); G, A, M, K, L: immunofixation for γ, α, μ, κ and λ chain, respectively.

d. 4 d. Isoelectric focusing/affinity-mediated immunoblotting analysis: Paraprotein bands did not react with Borrelia antigen but partly obscured CSF-restricted oligoclonal IgG pattern. Paired CSF and serum samples tested for (from left to right) "total" IgG, IgGκ, IgGλ; Anti-Borrelia IgG, IgGκ, IgGλ. Anode is at the top.

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(3) URINE ELECTROPHORESIS

1. The main reason for performing urine protein electrophoresis is to find a light chain myeloma producing an excess of free light chains (Bence Jones protein), an important part of a myeloma screen.

2. A band in the urine protein electropherogram may also result from an intact monoclonal immunoglobulin, especially if the patient has poor renal function.

3. Immunofixation is important in defining the nature of the band and in distinguishing between Bence Jones protein and an intact monoclonal protein originating from the serum.

4. From the urine electropherogram we can also tell if the proteinuria is of glomerular origin with a predominance of albumin, or if it has tubular components with excretion of smaller molecular weight proteins such as retinol binding protein and alpha-1 microglobulin.

5. Fragmented albumin in urine is occasionally seen but is of unknown significance. 6. Historically, urine has been concentrated by either removal of water from the specimen

leaving the proteins in higher concentration, or by centrifugation whereby the proteins are spun away from the majority of the water.

7. Demonstration of the protein components of urine from concentrated specimens was originally performed on cellulose acetate and later on agarose and high resolution agarose gel.

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(4) HEMOGLOBIN ELECTROPHORESIS Rationale:

1. Haemoglobin (Hb) abnormalities are the most frequent genetic disease, affecting approximately 7 per cent of the world population.

2. Today, abnormal Hbs are generally discovered during a systematic study performed within programmes for prevention of thalassaemias or sickle cell disease.

3. In several regions (India, Turkey, Irak, Iran, Gaza strip, Saudi Arabia, Cyprus, etc.), these are found during a premarital screening. In other regions, like west Europeans countries, the research for the main Hb disorders is often limited to populations at risk. It is done either as preconceptional or neonatal screening programmes.

Methods and patterns:

1. Hb S [β6 Glu>Val], the first abnormal Hb described, was discovered in 1949 by Pauling et al using moving boundary electrophoresis.

2. Zone electrophoresis performed on cellulose acetate strips (CAE) is still used in many clinical laboratories.

3. This technique has a resolution lower than that of IEF, but because of its simplicity remains among the more popular methods used in Hb screening. In this technique, the Hb molecules are separated at alkaline pH. Under these conditions all Hbs have a negative charge and migrate towards the cathode. Hb S, which has an additional positive charge compared to Hb A migrates more slowly.

4. In IEF, a pH gradient is established by carrier ampholytes submitted to an electric current and the Hb molecules migrate across this gradient until they reach the position where their net charge is zero (isoelectric point). The molecules will then concentrate in a sharp band as illustrated in Fig.

5. This technique allows separating molecules with isoelectric points differing only by 0.02 pH unit. Hb D-Punjab [β121 Glu>Gln], not detectable from Hb S by CAE, could easily be recognized by this technique. The disadvantage of this method is its relatively high cost and the requirement for a well-experienced laboratory staff.

6. This technique can help in diagnosing various hemoglobinopathies.

electrophoresis

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