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Electrophoresis
- 2. MOVING BOUNDARY ELECTROPHO RESIS / FRONTAL ELECTROPHO RESIS pH=7.6 Quantified optically by change in refractive index at the boundaries among these bands Homogeneous medium
- 3. pH=8.6
- 4. Factors affecting rate of migration Rate of migration (cm/s) per unit field strength (volts/cm) u = Q 6¶rn
- 5. Property of supporting media Cellulose Acetate Agarose Polyacrylamide
- 6. Temperature and Electrophoresis •During Electrophoresis -Denaturation of proteins -Smile effect -Heating effect of current and evaporation decreases resistance
- 9. Wick Flow Wick flow is due to flow of buffer in supporting medium due to evaporation of moisture from gel. It affect the migration of sample molecule.
- 11. In case of CSF, the endosmosis is set high to maximize the cathodal movement of immunoglobulins from the point of application over a span of the gel 1.For visual inspection 2. Detection of oligoclonal bands of immunoglobulin by optimizing the separation of the γ-region .
- 12. IONIC STRENGTH • Rate of migration is inversely proportion to ionic strength
- 13. General Operation Separation Detection Quantification
- 14. Lipoproteins - Sudan Black Lipoproteins for Oil Red O
- 15. Plasma Proteins - Amido black For plasma protein
- 16. Hemoglobins - Coomassie Brilliant Blue Ponceau S for hemoglobins
- 17. Ethidium Bromide (Fluorescent) Silver Nitrate for CSF Protein Sybr Green (Fluorescent dyes) for DNA Sybr Gold (Fluorescent dyes) for DNA
- 19. Paper Electrophoresis . Electrophoresis Tank-Inverted V Flynn and Mayo Type. Hanging strip inverted V type Covered tank divided in five compartments /Nylon strip
- 20. Horizontal strip type Paper taut in horizontal position Apparatus is wider than hanging strip type and buffer is farther apart Heating effect less marked due to smaller volume of atmosphere. Covered tank Whatman Nos.1 and 3MM.(thicker)
- 21. Shandon Scientific Company New Model Uses constant voltage and constant current supply,either of them can be selected by turning the switch. Uses 50-300 Volts at maximum 40mA as a constant current and 3- 40mA at maximum 300 Volts as a constant voltage
- 22. Shadon and Evans Electroselenium Model Horizontal strip type
- 23. Sample Applied:Along the pencil line suitably placed depending on direction of motion. In case of protien:Cathodal side of midline or apex of inverted V Quantitative Assesment:-Eluting( NaoH,acetic acid,Sodium carbonate) -Scanning Duration 8-10hrs .10 microlitre for 3cm ans 20 microlitre for 2 inch strip Buffer:Barbitone sodium barbitone (sodium dielthyl barbiturate and di ethyl barbituric acid) Stain :Bromophenol blue,Bromocresol green ,Lissamine green,Napthaline black
- 24. Starch Gel Electrophoresis Molecular seiving effect and better resolution Connecting at one end to a tray containing starch gel and at other end to electrode chambers Slot former (1.5x0.1x0.5cm) 6V/cm for 6 hr and current of 20-30mA.
- 26. After staining strips are made transparent by soaking in whitmore’s oil or liquid paraffin and then suitable for transmittance densitometry of the protein bands. 12x2.5 cm sample applied from micropipette or capillary along a line about 1 cm. on cathode side of midline Current:0.4 mA/cm width of paper. Dry and suspend in hot air oven at 80-100 0 C for atleast 10 min. Cellulose Acetate Electrophoresis . Its advantage over paper are- It has better resolving power,speed and ability to be cleared . No trobule of trailing and background after staining Washing is almost free from colour. Excellent seperation of plasma proteins (run-3-4 cm),taking not more than 2 hrs. Albumin and alpha1 bands are clearly distinguished. Disadvantage:Costly and greater skill is required to handle the Strips are brittle and dry.They are difficult to store 10-20 micro litres 6V/cm for 3hr Stain:PonceauS,Nigrosine
- 27. Agarose Gel Electrophoresis Principle and applications Agarose Gel Electrophoresis
- 28. Apparatus Gel Casting Trays Sample Combs
- 29. 120V and 7mA Stain:Amidoswartz 10 B Destaining agent:Methanol acetic acid
- 30. Agarose Gel Electrophoresis System 2-3 microlitre using pasteur pipette
- 32. SDS is an anionic detergent The sample is first boiled for 5min in buffer containing •Beta- Mercaptoethanol •SDS
- 34. Apparatus Glass Plates Casting Frame Gel Apparatus uffer Tank Comb Casting stand gels with frame
- 35. Application: •Serum protein electrophoresis •Hemoglobin electrophoresis •Lipoprotein electrophoresis •Enzymes (zymogen technique) •ALP isoenzyme electrophoresis -Separatin of smaller fragments of DNA -Separation of nucleic acids
- 37. • Cirrhosi Serum protein electrophoresis in cirrhosis
- 38. Protein electrophoretic patterns of serum (Ser)and concentrated urine (Ur) in a patient with nephrotic syndrome.
- 42. Serum and urine protein electrophoretic patterns in a patient with multiple myeloma. A predominance of the larger complete immunoglobulin. A large amount of the smaller-sized light chains with only a small amount of the
- 43. Transparent background Darkly stained fine arcs of antigen antibodies precipitates Stain:Amidoswartz 10 B,Nigrosine Stain Destaining agent:Methanol acetic acid,Ethanol water with trichloroacetic acid Kappa and Lambda can be obtained by immunodiffusion technique
- 44. Deficient immunoglobulin synthesis is revealed by a markedly diminished gamma band. Effected individuals are prone to recurrent infection
- 45. 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.
- 46. Bisalbuminemia. Serum protein electrophoresis with normal pattern in right lane and bisalbuminemia consisting of two equal intensity bands of albumin in left lane. The additional band of albumin in this patient migrated more slowly than normal albumin into the α1-region.
- 47. 2 Hemoglobin-Haptoglobin Complex type 1−1 Catalase (bubbles of O2) Type 2-2polymers Hemoglobin migrated deeply into the gel no band in the 1-1 position migrated as a series of high molecular weight polymers, each binding hemoglobin Type 1-2 haptoglobin minor band of 1-1 Series of high molecular weight polymers different from the bands observed with 2-2. in middle lane
- 48. Haemoglobin Electrophoresis Principle • At alkaline Ph (8.4-8.6), haemoglobin is a negatively charged protein and when subjected to electrophoresis will migrate toward the anode (+). • Structural variants that have a change in the charge on the surface of the molecule at alkaline pH will separate from Hb A. • Haemoglobin variants that have an amino acid substitution that is internally sited may not separate and those that have an amino acid substitution that has no effect on overall charge will not separate by • electrophoresis.
- 49. Note:Test sample should be compared with control sample containing known normal and abnormal haemoglobin. Control sample contains Hb A,F,S and C. Hb A,F,S and are always included in every electrophoresis. Sample:5 microlitre (Both test and control sample and covered with 50mm cover slip
- 50. Cathode (-) Origin…………………………………………………… ………….Carbonic anhydrase …………..A2' C…………A2, E, C-Harlem, O-Arab S…………..D, G, Q-India, Hasharon ……………Lepore .……………F A…………… ……………K-Woolwich J…………… ……………Bart's N………….. ………….. I …………... H Anode (+) • Schematic representation of relative mobilities of some abnormal haemoglobins. Cellulose acetate, pH 8.5.
- 51. Anode (+) C…………… S………………C-Harlem ………………Hasharon Origin…………. O-Arab, Q-India……………... A………………...D, E, G, Lepore, H, I, N, J F…………………Bart's, K-Woolwich Cathode (-) Schematic representation of relative mobilities of some abnormal haemoglobins. Agarose gel pH 6.0 using Tri sodium citrate dihydrate buffer using citrate agar.
- 52. Haemoglobin electrophoresis pattern from several different individual. Lane 1 and 5:Control. Lane 2:Normal Adult. Lane 3:Normal Neonate. Lane 4:Homozygous HbS individual. Lane 6 and 8:Heterozygous Sickle Cell individual Lane 7:Sickle Cell Disease individual.
- 55. Application: -Highly sensitive for studying the microheterogeneity of proteins . -Useful for separating the isoenzymes. -Research in enzymology, immunology. -Forensic, food and agriculture industry.,. -Human genetic lab
- 56. Isoelectric Focussing Electrophoresis Different gradient of the pH along the length of separating gel. Ampholyte:low molecular weight and varying isoelectric points
- 58. Two Dimensional Electrophoresis Can separate 1000 to 3000 proteins from the cell or an tissue extract.
- 59. Schematic representation of 2D Electrophoresis
- 60. High performance capillary electrophoresis(HPCE) /Capillary zone electrophoresis (CZE) /Free solution capillary electrophoresis(FSCE)/Capillary electrophoresis (CE)
- 61. Sample application is done by either of one method -High voltage injection -Pressure injection . Molecules are seperated on the basis of size,hydrophobicity and molecular mass
- 62. Positively charged molecule reach the cathode first (electrophoretic migration +electroosmotic flow). High voltage is applied (up to 50 kV) The components migrate at different rate along the length. Although separated by the electrophoretic migration, all the sample is drawn towards cathode by
- 63. Electro-osmotic flow DETECTION: near to cathode end, viewing window - Detected by the ultraviolet monitor, transmit signal and integrated by computer. -Refractive index -Fluorescence
- 64. Application: *Used to separate -Amino acids -Peptides -Proteins -DNA fragments -Nucleic acid -Drugs / even metals *Multiple myeloma testing (6bands). *Haemoglobinopathy screening. *Monitoring chronic alcoholism (GGT). *HbA1c
- 65. Isotachophoresis • Used for separation of smaller ionic substances. • They migrate adjacent with contact one another, but not overlapping. • The sample is not mixed with the buffer prior to run. • Hence current flow is carried entirely by the sample ions. • Faster moving ions migrate first and the adjacent ones next with no gap between the zone . • All ions migrate at the rate of fastest ion in zones. • Then it is measured by UV absorbance Separation of small anions and cations Amino acids Peptides Nucleotides Nucleosides Proteins.
- 66. Pulsed Field Electrophoresis • Power is alternately applied to different pair of electrodes/ electrode arrays, so the electrophoretic field is cycled between two directions. Electrophoretic field is cycled at 105-1800 Because of which the molecule have to orient to the new field direction . This permit separation of large molecule like DNA . Applied: For typing various Strain of DNA.
Hinweis der Redaktion
- Every aminoacid has its own specific isoelectric point. At pH above it isoelectric point,protein will have net negative charge and will migrate toward anode. Since prealbumins isoelectric point is far away from buffer pH, it is expected to have greater charge and migrate fastest toward anode. Gamma Globulin :Isoelectric pont is closest to buffer Ph has least charge and migrates toward anode.
- We applied a sensitive silver nitrate stain to SDS-polyacrylamide gels to detect oligoclonal bands in unconcentrated CSF. Silver staining took less than 3 hours and gave easily readable bands in 93% of MS patients. In contrast, standard Coomassie Blue stain was positive in only 53%. Maximum resolution and numbers of bands resulted when silver staining was used on 10 to 20 microliters of CSF. Silver staining of SDS-polyacrylamide gels permits reliable oligoclonal band detection on small volumes of unconcentrated CSF.
- Paper is cut into strips, the from the remainder of the paper as blank.is eluted by appropriate solvent and the colour of resulting solutions read in an absoptiometer using a strip . -Scanning: Strip is made transluscent by putting oil in it. And placed in scanner and graph is plotted. Unoiled strip may be directly scanned in an instrumentwhich measures reflected light to give tracings. The size of areas below each setionof curve can be measured by planimeter and percentage of fraction contibued is determined
- Prealbumin is defined electrophoretically as the fraction that migrates in a position faster than albumin toward the anode
- 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
- Phenotyping of haptoglobin. Patient serum specimens were mixed with free hemoglobin from erythrocytes lysed in vitro and electrophoresed in a polyacrylamide gel with h3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide. Hemoglobin exhibits pseudoperoxidase activity that converts substrigh resolution of different molecular weight proteins. After electrophoresis, the gel was developed in a solution of substrate to a blue product wherever it has migrated including complexes with haptoglobin. Bubbles of oxygen formed in the gel at the location of catalase, which is also present in the lysate. Type 1-1 haptoglobin (arrow in left lane) migrated deeply into the gel (from top to bottom), although unbound hemoglobin migrated even farther. Type 2-2 haptoglobin showed no band in the 1-1 position but migrated as a series of high molecular weight polymers, each binding hemoglobin (bar next to right lane). Type 1-2 haptoglobin (middle lane) showed a minor band of 1-1 plus a series of high molecular weight polymers different from the bands observed with 2-2.
- A positive solubility or sickling test indicates the presenceof Hb S and as such is useful in the differential diagnosis of Hbs D and G, which migrate with Hb S on cellulose acetateelectrophoresis at alkaline pH. Positive results are also obtained on samples containing the rare haemoglobinsthat have both the Hb S mutation and an additional mutationin the b chain. A positive solubility test merely indicates the presence of a sickling haemoglobin and doesnot differentiate between homozygotes, compound heterozygotes and heterozygotes. In an emergency, it may benecessary to decide if an individual suffers from sickle celldisease before the haemoglobin electrophoresis results areavailable. In these circumstances, if the solubility test ispositive, a provisional diagnosis of sickle cell trait can be made if the red cell morphology is normal on the blood film. If the blood film shows any sickle cells or numerous target cells, irrespective of the Hb, a provisional diagnosis of sickle cell disease should be made; many patients with sickle cell/Hb C compound heterozygosity will have a normal Hb. Remember that the sickle test is likely to be negative in infants with sickle cell disease. False-positive results have been reported in severe leucocytosis; in hyperproteinaemia (such as multiple myeloma);and in the presence of an unstable haemoglobin, especially after splenectomy. The use of packed cells, as described in this method, minimizes the problem of false-positive results caused by hyperproteinaemia and hyperlipidaemia.False-negative results can occur in patients with a low Hb and the use of packed cells will overcome this problem.False-negative results may also occur if old or outdatedreagents are used and if the dithionite/buffer mixture is not freshly made. False-negative results are likely to be found in infants younger than age 6 months and in other situations (e.g. post-transfusion), in which the Hb S level is <20%. All sickle tests, whether positive or negative, must be confirmed by electrophoresis or HPLC at the earliest opportunity.
- Interpretation and Comments Figure 14.3 shows the relative electrophoretic mobilities ofsome common haemoglobin variants at pH 8.5 on cellulose acetate. Satisfactory separation of Hbs C, S, F, A andJ is obtained (Fig. 14.4). In general, Hbs S, D and G migrate closely together, as do Hbs C, E and OArab. Differentiationbetween these haemoglobins can be obtained by using acid agarose gels, citrate agar electrophoresis (pH-6), HPLCor IEF. However, there are slight differences in mobility between Hbs S, Lepore and DPunjab and also between HbsC and E; optimization of the technique will facilitatedetection of the difference. Generally, the Lepore Hbs and Hb DPunjab migrate slightly anodal to Hb S (i.e. they are slightly faster than S); Hb C migrates slightly cathodal to Hb E (i.e. it is slightly slower than E).All samples showing a single band in either the S or C position should be analysed further using acid agarose or citrate agar gel electrophoresis, HPLC or IEF to exclude the possibility of a compound heterozygote such as SD,SG, CE or COArab. The quality of separation resulting from this procedure is affected primarily by both the amount of haemoglobin applied and the positioning of the origin. Also, delays between application of the sample and commencement ofthe electrophoresis, delay in staining after electrophoresis or inadequate blotting of the acetate prior to applicationwill cause poor results. This technique is sensitive enough to separate Hb F from Hb A and to detect Hb A2 variants.If an abnormal haemoglobin is present, the detection of a Hb A2 variant band in conjunction with the abnormal fraction is evidence that the variant is an a chain variant. Globin electrophoresis at both acid and alkaline pH isalso useful in elucidating which globin chain is affected. However, with the more ready availability of HPLC, it is less often needed.When an abnormal haemoglobin is found, it may be of diagnostic importance to measure the percentage of the variant; this can be done by the electrophoresis with elution procedure for Hb A2 estimation given on p. 322.Quantitation of Hb S is often clinically useful, both inpatients with sickle cell disease who are being treated by transfusion and for the diagnosis of conditions in which Hb S is coinherited with a and b thalassaemia, as outlined in Table 14.5. Quantitation of Hb S can be done withHPLC, electrophoresis with elution or by microcolumn chromatography.
- Sicke cell beta thalassemia Hb F, S, A2.
- The isoelectric point (pI, pH(I), IEP), is the pH at which a particular molecule carries no net electrical charge. Biological amphoteric molecules such as proteins contain both acidic and basic functional groups. Amino acids that make up proteins may be positive, negative, neutral, or polar in nature, and together give a protein its overall charge. At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge. Proteins can, thus, be separated by net charge in a polyacrylamide gel using either preparative gel electrophoresis or isoelectric focusing, which uses a pH gradient to separate proteins. Isoelectric focusing is also the first step in 2-D gel polyacrylamide gel electrophoresis. These molecules migrate to their respective isoelectric points when a current is applied, resulting in a pH gradient being formed; for haemoglobin analysis, a pH gradient of 6–8 is usually used. Haemoglobin molecules migrate through the gel until they reach the point at which their individuals isoelectric points equal the corresponding pH on the gel. At this point, the charge on the haemoglobin is neutral and migration ceases. The pH gradient counteracts diffusion and the haemoglobin variant forms a discrete narrow band.
- Electro-osmosis: When the voltage is applied to the circuit, one electrode become net positive and the other net negative. The (wall’s) immobile silanol anions pair with mobile buffer cations, forming a double layer along the wall (wall--> buffer cations-->buffer anions-->bulk buffer solution). The remaining buffer cations are attracted to the negative electrode, dragging the bulk buffer solution with them. This is electroosmotic flow. For an uncoated capillary, the EOF is toward the negative electrode.
- DIFFERENT MODES OF OPERATIONS Separation principle Capillary zone electrophoresis Charge to mass ratio of molecule Micellar electrokinetic chromatography The separation is accomplished by micelles formation. (8-9mmol/L for SDS) -During migration, micelle interact with analyte as chromatographic manner and the separation is brought about. Used for separation of the neutral and charged solutes. Capillary gel Electrophoresis Seperation based on the sieving. The capillary is filled with “sieving matrix” or “soluble polymer network” Cross linked polyacrylamide- choice of polymer. Variety of polymeric matrices are available for DNA and Protein. Capillary isoelectric Focussing Electrophoresis Is comparable to tube IEF. -Carried out in the capillary. -The focused zone migrate to the detector with the separated sample. -cIEF is completed in ~15 min. Capillary Isotachophoresis Same feature as ITP. -Except condition of pure ITP not achieved. -Typically used for online sample preconcentration.