Purification And Quantification Of Immunoglobulin G: Using Electrophoresis, Chromatography, And Bca.

Purification and Quantification of Immunoglobulin G: Using Electrophoresis, Chromatography, and BCA.

The immune system protects the organism against foreign and potentially noxious entities. Immunoglobulins are part of the adaptive response. IgG is the principal antibody in blood and extracellular fluids, it accounts for 75% of the immunoglobulins present in blood, IgG can also activate thecomplement system. We utilized various steps to purify and quantify IgG. The technique used for purification was affinity chromatography; this technique involves using a gel that contains protein A and agarose (sefarose), this protein is found in Staphylococcus aureus. We also ran two electrophoresis, the first was an agarose gel electrophoresis done using serum as out analyte of interest, thispresented an overview of the serum's composition. The second was an SDS-PAGE, done to observe the presence of IgG in the serum. The agar electrophoresis established the serums components: Albumin, and globulins [alfa1 (α1), alfa2 (α2), beta1 (β1), beta2 (β2) y gamma (γ)]. The affinity chromatography provided the division between IgG and the other proteins in the serum. The BCA analysis provided theconcentrations of IgG in the serum, it also provided the concentrating of other proteins, these proteins were not identified. Afterwards a SDS-Page was run to confirm the presence of IgG. After the whole process was completed the concentration of IgG in our sample was too small to be distinguished in the polyacrilamide electrophoresis. The results form the BCA test confirm the presence of IgG itsconcentration was very low, the absorbance fell below zero after the statistical analysis.

The immune system protects the organism against foreign and potentially noxious entities. The immune system contains the innate response and the adaptive response. The innate immune system consists of several lines of defense that the body contains to protect itself against pathogens; these defenses arethe same no matter what the pathogen is. The innate response is always the same, and sometimes it is not enough to stop the pathogen. When the innate response is unable to stop the pathogen the adaptive response is activated. The adaptive response is composed of a wide diversity of methods to stop pathogens; for the purpose of this experiment we will focus on immunoglobulin (Ig), specifically IgG.Immunoglobulins are also referred to as antibodies. Antibodies are found in the fluid component of blood, or plasma, and in extracellular fluids. Because body fluids were once known as humors, immunity mediated by antibodies is known as humoral immunity. The B- lymphocytes are the primary cells responsible for the recognition of antigens. Immunoglobulin G is expressed on the membrane of theB-Lymphocyte or it is secreted by the B cell. Antibodies are glycoproteins, composed by four polypeptide chains, two heavy chains and two light chains. The heavy chains are identical, about 445 amino acids (60kDa), and the light chains are about 214 (25kDa). After the chains interact they attain a Y form. Part of the heavy chains and the light chains form the arms of the antibody, while the remainingpart of the heavy chains form the base of the antibody (this part may form part of the cell's membrane). The arms of the structure are known as the variable region, and the base is known as the constant region. The variable region is the one that bonds to the antigen. Immunoglobulins are divided into four classes IgM, IgD, IgG, IgA, and IgE. The class will vary depending on the amino acid sequencereflected on the constant region. IgA is usually found in saliva and maternal milk. IgE is important in the event of a parasitic infection, or in allergic reactions, they provide the necessary chemicals like histamine.3 IgM is the first immunoglobulin that B cells express in their membrane, they form pentamers when they are secreted into the blood stream. IgD is found in virgin mature B cells;...