Electrophoresis Part 2 PDF

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Summary

Factors the Influence Rates of MigrationElectroendosmosis- The movement of buffer across the support medium when an electric field is applied. - The negatively charged proteins can create their own negative ion clouds. The agarose gel has fixed hydroxyl anions, that don’t move, from water inside the...

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MLSC-3111, Clinical Biochemistry II Factors the Influence Rates of Migration Electroendosmosis -

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The movement of buffer across the support medium when an electric field is applied. The negatively charged proteins can create their own negative ion clouds. The agarose gel has fixed hydroxyl anions, that don’t move, from water inside them. This forms a positive ion cloud at the surface of the gel when those hydroxyl anions attract positive ions from the buffer. The buffer’s positive ion cloud on the surface is attracted to the negative cathode of the electrophoretic system and migrates toward it. This creates an osmotic flow which pulls the buffer water towards the cathode. The proteins, which are supposed to go towards the positive anode, are also pulled by the osmotic flow in the opposite direction. Therefore, protein molecules are being pushed back from a “wave” of buffer moving in the opposite direction. Uncharged and weakly charged macromolecules are washed along with the buffer water flow towards to cathode instead of the anode. The gamma globulin band in bodily fluids will separate more sharply by being pushed to the cathode and will appear behind the point of sample application in some systems. The gel attracts positive ions from the buffer water to create a positive cloud that migrates toward to negative cathode with the proteins. Causes a chemical change.

Wick Flow -

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Caused by the movement of buffer into the support medium. Heat generated during the reaction will cause moisture evaporation from the centre of the gel because the ends are immersed in the buffer with a lot of water while the middle is not. Naturally, the water at the end will flow into the centre from both sides to replace the evaporated buffer. This causes an equal flow of buffer from both sides to the centre which will affect the migration of proteins. The flow is directly proportional to the rate of evaporation and the flow of the solvent will be higher at the ends compared to the centre of the support. This can be offset by placing the sample application on the side that allows migration in the same direction as the wick flow. Can also cover the system to reduce evaporation. The heat from electrophoresis dries the gel in the middle to draw buffer to dry areas. May cause bands to be distorted or atypical in shape. This causes a physical change.

MLSC-3111, Clinical Biochemistry II Other Applications of Electrophoresis -

Hb Electrophoresis, Hb are sorted based on size and shape just like proteins. Lipoprotein Electrophoresis, performed to determine risk for cardiovascular disease. Detects Chylomicrons, LDL, VLDL and HDL. Isoenzymes, include… o LD, found in many tissues. o ALP, found in liver and bone. o CK, found in heart, skeletal muscle, and brain.

Other Electrophoresis Information -

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Sample Application, successful electrophoresis requires application of correct amount of sample with clean applicators and no air bubbles. Fixation, some protein bands in electrophoresis need additional fixation so they don’t wash off during staining. This can be done with agarose gels and Paragon Blue dye, heat or precipitating proteins into medium using saline. Stains, used to visualize separations after gel is washed and fixed. After staining, excess stain is washed away. The background may need to be cleaned of stain using CAE solvent to clearly see the bands.

Trouble Shooting Atypical Bands -

Short Migration Patterns, can be caused by low voltage. Unequal Migration Rates, can be caused by dirty electrodes, dried areas of gel, uneven gel surfaces, etc. Distorted Bands, can be caused by air bubbles, inadequate blotting or poking the gel. Weak Bands, can be caused by too little sample applied, not enough stain used or using a diluted stain.

Electrophoresis Detection -

Detectors, required to convert visual separations to quantitative tracings after support medium had been dried and stained. Densitometry, measurement of the absorbance of analytes along the length of a support. Uses a densitometer which is a modified spectrophotometer.

Densitometer -

Stained strip is scanned using optimal wavelength of light. Light is transmitted through to the photodetector that senses the change in transmittance due to density of protein fraction.

MLSC-3111, Clinical Biochemistry II -

The amount of light transmitted is proportional to the concentration of protein in the fraction. This provides a plot of absorbance versus distance of strip as it passes the optics reader in the form of a tracing or graph.

High Resolution Scanners -

Uses a flat-bed scanner that scans in a picture of the electrophoretic gel and creates an electronic plot of gel. Uses reflectance spectrophotometry.

Digital Image Analyzers -

Camera used to scan and store digitized light intensity readings from large areas and performs a plot of the gel.

Components of a Densitometer -

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The densitometer creates electrophoretograms using the absorbance measurements from the strip. The densitometer displays the bands as peaks in absorbance on the graph. Quantitation of proteins is done through integration when the area under the curve is measured at each peak. Using this, a percentage can be generated when it is compared to the total protein value. % Protein Fraction x TP (g/L) = Protein Fraction (g/L). Each peak is individually quantitated and routine serum protein electrophoresis (SPE) reports 5 fraction concentrations. High Resolution SPE, separates beta into beta 1 and 2 peaks and can report 6 fractions. These kits are more costly so a 5 fraction SPE is typically done as it is sufficient enough for diagnosis.

Additional Types of Electrophoresis Capillary Electrophoresis (CE) -

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Electrophoresis that takes place in a very small, thin fused silica capillary tube lined with agarose gel. The ends of the tube are placed into a buffer reservoir on each end and then applied with an electrical field. The capillary acts as an electrophoretic chamber itself. It has a gel lining, a buffer running through it and a charge running through it. Electroosmotic flow has a huge effect on this type of electrophoresis. Electroendosmotic Flow (EOF), the fundamental concept of CE. Same principle as electroosmosis. The EOF is a stronger force than the electrical field in this situation, causing all proteins to be carried away toward to the cathodic end of the capillary. This flow is superimposed on electrophoretic migration.

MLSC-3111, Clinical Biochemistry II -

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Cations migrate fastest along with osmotic flow (positive ions are attracted to the negative cathode). Neutral molecules are carried by the EOF to the cathode next since they have no preference of charge. Anions migrate the slowest because they are already negatively charge and will be repelled by the cathode. Furthermore, weakly charged anions will move the slowest. CE uses the highest voltage possible with the shortest time to produce high resolution separation. The high voltage produces a lot of heat, but capillaries will dissipate the heat away. However, the CE does not produce a permanent film that can be visualized. The proteins migrate through the capillaries detector window and then go straight into the waste. Uses UV light for detection.

Immunofixation Electrophoresis (IFE) -

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Following an initial normal electrophoresis, antiserum against the protein of interest is spread directly onto the gel. The protein of interest binds to the antiserum and precipitates into the gel matrix (immunoprecipitation technique). The gel is then washed out leaving behind the antiserum-protein complexes in positive reactions for the protein of interest. The method is qualitative as bands are not quantitated. Used in patients suspected of Multiple Myeloma. IFE is a two stage procedure. o Stage 1, the sample is diluted to bring abnormal Ig into a concentration that reacts best with the reagent antibodies. The diluted sample is then applied to all 6 tracks and then electrophoresed. The applied sample migrates equally in all tracks. o Stage 2, a fixative is added to track one. Corresponding antisera is added to each track to react with the protein of interest (IgG, IgA, IgM, kappa, and lambda antisera added. One Ab type per track). The gel is the washed, stained destained for background stain and then dried for viewing. Fixative is used in track one because we want that track to stay for comparing to the other tracks. If we didn’t wash the gel we would have other proteins sticking all over the place and interfering with viewing the complexes. A normal serum sample will exhibit no stain or just background staining. No protein means no precipitation into the gel and nothing to stain. A sample with abnormal protein will exhibit banding at the same migration location across all tracks containing antisera. Banding results from precipitation with antisera allowing the ID of the protein.

MLSC-3111, Clinical Biochemistry II Protein Patterns in SPE -

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Proteins are separated into 5 fractions: Albumin, alpha-1, alpha-2, beta and gamma globulin. The point of the origin is close to the negatively charged cathode and the albumin fraction has an isoelectric point that differs the most from the pH of the gel. This causes it to have the highest charge to mass ratio, making it the farthest/fastest moving protein from the cathode toward the anode. The albumin band is the only homogenous band on the SPE gel. All the other bands are heterogenous. Prealbumin, now known as transthyretin. Not seen on SPE and is found mainly in CSF. It is a marker for nutritional status and is measured in other assays. Has a short half-life and is the first protein to be lowered in malnutrition. Albumin, smallest and highest net charge of bands detected on routine SPE. It migrates the fastest and farthest to the anode and its peak is homogenous. Alpha-1 Globulin, 90% of it is alpha-1-antitrypsin. o Alpha-1-antitrypsin, Inhibits proteolytic enzymes and a lack of it causes trypsinlike enzymes to not be neutralized. Leads to tissue damage and emphysema. o Alpha-fetoprotein (AFP), major fetal plasma protein that rapidly disappears after birth. Elevated in liver disease (hepatitis and cirrhosis) and is a cancer marker for liver and testicular cancer. Very high in spinal bifida. Alpha-2 Globulin, has 12 peak components. Hemoglobin from hemolysis is complexed to haptoglobin and will appear as a small band in the late alpha-2 zone. The sixth fraction that may occur is due to this and should not be reported as an abnormal protein. Hemolyzed samples need recollection. o Ceruplasmin (CER), aids in the diagnosis of Wilson’s Disease. Patients exhibit copper deposition in the cornea (Kayser-Fleischer rings). o Alpha-2 Macroglobulin (AMG), elevated 10-fold in nephrotic syndrome and mildly in diabetic nephropathy. o Haptoglobin (HAP), aids in of in-vivo hemolysis. Also lowered in liver disease. Beta Globulin, characterized by an “M” peak on the electrophoresis graph. Fibrinogen migrates to the beta fraction to cause an extra band known as beta-gamma. True serum will not contain any fibrinogen, but plasma will show this band on the EPS. o Transferrin, transports iron. o Beta-Lipoprotein, transports lipids. o Complement, lyses foreign cells. o Fibrinogen, coagulation factor. Seen as a separate band between the B and G regions on SPE. Gamma Globulin, slowest moving protein and migrates near the cathode. Peak components include immunoglobulins and C-Reactive protein....