Quiz 5 Study Materials Fall 2024 PDF

Biol 1002 – Fall 2024 – Instructor Dr. Polle Quiz 5 - Selected study materials from Lab 11 Western Blotting Western blotting is an immunodetection technique used by proteomic scientists to detect and quantify specific proteins in complex biological samples. Proteins ere then transferred or "blotted...

Biol 1002 – Fall 2024 – Instructor Dr. Polle Quiz 5 - Selected study materials from Lab 11 Western Blotting Western blotting is an immunodetection technique used by proteomic scientists to detect and quantify specific proteins in complex biological samples. Proteins ere then transferred or "blotted" from the gel onto a paper-like membrane. A specific antibody, engineered to bind only to the protein of interest, is added to the membrane. This antibody is attached to a compound that causes a colored reaction, enabling scientists to detect and quantify a single protein of interest from hundreds of other proteins in a sample with high accuracy. Subunit of a myosin light chain from the many thousands of proteins comprising the muscle tissues of different fish. Myosin light chain proteins will be compared from different species for variation, commonality, or evolutionary divergence. What is the purpose of the actin & myosin standards? The molecular mass of myosin light chain 1 is approximately 20 kD, myosin heavy chain is 200 kD and actin is 42 kD. Which proteins will migrate fastest through the gel? Why? What was the purpose of the Coomassie stain? Proteins are electrophoretically transferred from the gel onto a nitrocellulose membrane. Proteins, still negatively charged from the SDS, migrate out of the gel and bind to the surface of the membrane, creating a mirror image of proteins separated in the original gel. Recall the assembly of the “sandwich” for Blotting. Nitrocellulose membranes: Nitrocellulose acts as a solid support for proteins bound to its positively charged surface. These durable membranes can undergo multiple wash and incubation steps, and provide a white background on which to visualize the color development at the site of the protein of interest only. Biol 1002 – Fall 2024 – Instructor Dr. Polle Why do proteins migrate from the gel to the nitrocellulose membrane? Recall the need for antibodies for detection of proteins! What does the primary antibody do? Why did we use a secondary antibody? Be able to describe how a specific protein can be identified from a mixture of proteins. Selected study materials from Lab 12 GMO, DNA isolation, and PCR - Be able to define what the term GMO means. - What is the gene for which protein is often used to make GMO crops. - How would you make a GMO plant? - Recall that the objective of PCR is to produce a large amount of DNA in a test tube (in vitro). - Recall the steps of DNA extraction and why these steps were performed as well as why certain components were used in the isolation procedure. - Be able to describe the DNA isolation process in detail. - Be able to explain why PCR is used to determine if food is derived from a GMO? Biol 1002 – Fall 2024 – Instructor Dr. Polle - Recall what PCR is, what the reagents are and what the steps within the cycle accomplish. - Recall that PCR involves a repetitive series of cycles, each of which consists of template denaturation, primer annealing, and extension of the annealed primer by Taq DNA polymerase. - Ensure that you understand the answers to all questions about GMO, PCR and gel electrophoresis in your laboratory 12 and 13 manual. - Be able to explain what primers are. - Why were two different primer pairs used in Laboratory#12?