module 3

• Three Centrifugation methods are Differential pelleting, Rate zonal, and Isopycnic.
• Differential pelleting involves lysing the cells to expose their contents, centrifuging at various RPMs, and removing the supernatant.
• At the lowest RPM, heaviest organelles such as nucleus form sediments, while at highest RPMs, lightest particles like ribosomes float to the top.
• Rate zonal centrifugation involves using different concentrations of sucrose to form a density gradient, putting in the sample, and centrifuging so the samples settle in layers which equal their density.
• Isopycnic centrifugation separates particles based on their buoyant density, filling a tube with gradient forming substance, putting in the sample, and centrifuging.
• Gel filtration (Gel permeation chromatography) separates molecules based on size, using a column with sephadex beads, putting the sample on top, and measuring elution volume.
• Anion exchange chromatography separates particles, such as proteins, based on their charge, putting a positively charged bead, putting the sample on top, and measuring elution volume.
• Affinity chromatography uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of interest, setting up the samples, and measuring elution volume.
• B cells produce antibodies.
• Immunohistochemical staining, Immuno-fluorescent staining, and ELISA are methods of staining antibodies.
• There are 3 types of enzyme linked immunosorbent assay: Direct assay, Indirect assay, and Sandwich ELISA.
• In a Direct ELISA, the primary antibody binds directly to the antigen.
• In an Indirect ELISA, the primary antibody binds to the antigen, and then a secondary antibody binds to the primary antibody.
• In a Sandwich ELISA, the antigen is pinched between 2 antibodies.
• To find misfolded proteins, homogenize a sample of brain tissue, digest with Proteinase K, denature proteinase K, and perform ELISA, with misfolded protein as antigen.
• The pregnancy test involves a sample entering the reaction zone, the hCG binding to the 1st antibody, which has an enzyme attached to it, then going to the test line, and if hCG is present it will bind to the 2nd antibody.
• SDS is used in SDS-PAGE to neutralize the effect of protein folding and charge on speed of migration.
• If no lines are present in a pregnancy test, it is considered faulty.
• Ion exchange chromatography can be used for desalination, removal of contaminants, and purification of proteins.
• Isoelectric focusing (IEF) is an electrophoresis through a stable pH gradient such that a charged molecule migrates to a position corresponding to its isoelectric point.
• RAT test works like a pregnancy test, just detects virus antigen instead of hCG hormone.
• Cation exchange should be used for positively charged protein as the buffer is negative and will hold onto positively charged proteins.
• pI is the pH at which protein has no charge.
• SDS-PAGE is used for separation of proteins according to size, and small DNA fragments.
• Proteins have positive charge at low pH, negative at high pH.
• Differences between molecules used to separate them include charge, size, shape, density, and affinity.
• Proteins bands are visualized in SDS-PAGE using Coomassie blue or transillumination.
• Anion exchanger is positively charged, attracts negatively charged anions, use buffer with positive charge.
• Cation exchanger has negative charge, attracts positively charged cations, use buffer with negative charge.
• Discontinuous buffer structure in SDS-PAGE includes stacking gel with low polyacrylamide content, allowing for same proteins to concentrate into one band, and different proteins to effectively separate into different bands, and separating gel with higher polyacrylamide content and higher pH.
• Western blotting steps include SDS-PAGE, transfer onto nitrocellulose membrane, specific antibodies binds to the protein of interest, incubate with secondary antibody that carries a tag for detection purposes.
• The 1st antibody, bound or unbound to hCG, continues to the control line, where the 1st antibody binds to a 3rd antibody, again realizing an enzyme which forms a dye.
• If both lines show in a pregnancy test, it indicates that hCG is present, and pregnancy has occurred.
• If the control line is present in a pregnancy test, it indicates that there is no hCG present, indicating no pregnancy.
• SDS-PAGE stands for SDS polyacrylamide gel electrophoresis.
• Anion exchange should be used for negatively charged protein because the buffer has positive charge and can hold onto the negatively charged protein.
• Proteins become negatively charged at high pH and positively charged at low pH.
• Sedimentation speed depends on densities of medium and solvent, with higher solvent density and lower medium density meaning a faster rate of sedimentation.
• The protein module in BIOL2103 includes techniques such as antibody technique, chromatography, and electrophoresis.
• Lab technician in the protein module is responsible for techniques such as antibody technique, chromatography, and electrophoresis.