1. Biochemical instrumental analysis-7 Dr. Maha Al-Sedik

3.  Atoms are neutral because the protons which are positive and electron that are negative balance out the charge, so there are same numbers of protons and electrons in an atom.  An ion is an atom or group of atoms that has a positive or negative charge.  Ion is formed by losing one or more electrons or gain one or more electrons from their highest occupied energy levels.  There are two forms of ion, cation and anion.

10. Ions are atoms which have gained or lost one or more electrons giving the ion a net positive or negative charge.

11.  Cation positively charged ion, it moves toward the cathode (-).  Anion negatively charged ion, it moves toward the anode (+).  Amphoteric substance can have a positive or negative or zero charge, it depends on conditions.

12.  Proteins are amphoteric compounds as they contain both acidic and basic residues.  Each protein has ionizable groups,amino or carboxyl groups, in solution they give: COOH + HOH COO - + H 3 O NH 2 + HOH NH3 + + OH

13.  The overall charge of the protein is determined by the number of acidic and basic amino acids in its basic structure. Because of their amphoteric nature, amino acids can express a net positive charge, a net negative charge or a net charge of zero.

14. At one pH, called the isoelectric point, the number of positive and negative charges are equal. At this pH, the protein exhibits a net zero charge, and is referred to as a ‘zwitterion’. At this point the protein will not migrate in an electrical field and remains at application point. Every amino acid has its own specific isoelectric point.

15. When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.

16. Electrophoresis Electrophoresis is the process of moving charged biomolecules in solution by applying an electrical field across the mixture. Electrophoresis Electrophoresis is a separation technique based on the principle that a charged particle in solution will migrate towards one of the electrodes when placed in an electrical field.

17. Electrophoresis is a technique used in clinical laboratories to separate particles (proteins) from each other:  Proteins in body fluids: serum, urine, CSF.  Proteins in erythrocytes: hemoglobin.  Nucleic acids: DNA, RNA.

18. Factors influenced electrophoresis mobility:  The electric field.  Net charge of the molecule.  Size and shape.

19. Instrumentation and reagents:  Two buffer boxes contain the buffer used in the process.  Each buffer box contains an electrode made of either platinum or carbon, the polarity of which is determined by the mode of connection to the power supply.  The electrophoresis support on which separation takes place may contact the buffer directly, or by means of wicks.  The entire apparatus is covered to minimize evaporation and protect the system.  The power supply to provide electrical power.

20. In most electrophoresis units, the gel is mounted between two buffer chambers containing separate electrodes so that the only electrical connection between the two chambers is through the gel.

22. General operations performed in conventional electrophoresis include: General operations performed in conventional electrophoresis include: (1) separation (2) staining (3) detection (4) Quantification

23. Buffers Function of buffer: Function of buffer: 1. Carries the applied current. 2. Established the ph.

24. At one pH, called the isoelectric point, the number of positive and negative charges are equal. At this pH, the protein exhibits a net zero charge, and is referred to as a ‘zwitterion’. At this point the protein will not migrate in an electrical field and remains at application point. Every amino acid has its own specific isoelectric point. At a pH above its isoelectric point, the proteins will have a net negative charge and will migrate towards the anode.

25. The speed and direction a charged particle moves is determined by: Factors related to particle:  Size and shape.  Net charge (determined by buffer pH). External factors :  Voltage.  Buffer pH.  Support medium (type of gel).  Temperature:↑speed but ↑↑ leads to denaturation of protein.

26. Electrophoresis of plasma protein  The proteins found in plasma have amino acids as their subunits, and each protein has its own specific isoelectric point.  Because of their different isoelectric points, each protein will move at a different rate when placed in an electrical field.  Proteins with similar isoelectric points will migrate to a similar area in an electrical field.

27. o Normal total plasma protein concentration: 6 – 8.5 gm / dl. o Electrophoresis separates serum Proteins into 5 distinct zones or bands at barbital buffer at pH 8.6 :  Albumin.  Alpha globulin ( Alpha 1 and Alpha 2 ).  Beta globulin ( Beta 1 and Beta 2 ).  Gamma globulin: IgG, IgA, IgM, IgD, IgE and C-reactive protein.

28. The width of each band is dependent upon the number of proteins that are present in that fraction.

29. Electrophoresis separates TSP into 5 distinct zones or bands at ph 8.6:  Albumin  Alpha-1 globulin.  Alpha-2 globulin.  Beta globulin.  Gamma globulin: IgG, IgA, IgM, IgD, IgE and C-reactive protein.

30. Q – WHAT is the difference between serum proteins and plasma proteins in electrophoresis?

31. Serum: is a clear yellowish fluid that remains from blood plasma after clotting factors (fibrinogen, prothrombin ect.) that have been used in the formation of a clot. Plasma: is a clear yellowish fluid that still contains all of the clotting factors and have not been solidified into clot.

32. If plasma is used in electrophoresis, there will be an extra band ( fibrinogen ) between beta and gama bands. Fibrinogen

33. Basic Procedure:  Sample is applied to an agarose gel.  Gel is placed into electrophoresis cell.  containing barbital buffer at pH 8.6.  Power is applied creating an electrical field and the proteins are separated.  Proteins are fixed to the gel and stained.  Separated proteins on gel are scanned.