NE102 Lecture Notes 2

2 membranes are “permeablized” with a

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Unformatted text preview: 2. Membranes are “permeablized” with a detergent (e.g., Triton X-100) a. Punching holes in the membrane to allow the antibodies in. 3. Cells are incubated wit the antibody conjugated to a fluorescent molecules. 4. View it with a fluorescent microscope. Antibodies as Probes for Proteins, pt. 2 19:00 Antibodies can also be used to examine RELATIVE AMOUNTS of a protein of interest. Could be done by immunocytochemistry, but more often done by immunoblot . IMMUNOBLOT (Western blot) What is immunoblot? Method by which proteins in a cell lysate are immobilized and detected on a membrane Mixture of proteins put through SDS-APGE Transfer to filter/membrane Incubate filter with antibody against protein of interest Antibody binds to specific protein (Antibodies can be labeled with luminescent molecules) Take a piece of film and areas of light will turn black Immunoblotting is semi-quantitative The amount of antibody bound to the immunoblot will be PROPORTIONAL to the amount of antigen Ex.) 100 molecules = 100 antibodies Immunoblot can be used to examine changes in protein levels over time IMMUNOPRECIPITATION (aka IP) “Co-IP” can detect protein-protein interactions Translation and Translational Regulation 19:00 Reminders: In translation, the nucleotide sequences is read in triplets = codons Each codon codes for a specific amino acid Codons are read 5’ to 3’ to direct polypeptide synthesis amino to carboxy terminus AUG = start codon – encodes MET UAA, UAG, UGA = stop codons New point: The 5’ and 3’ ends of mRNA that are not translated into proteins are called 5’ & 3’ untranslated regions ( 5’ UTR & 3’ UTR ) tRNAs serve as “adaptors” between codons and amino acids Brings the anticodon that complementarily pairs with the codon sequence OVERRIDING QUESTION: How does the process of translation take place? Important prerequisite = attaching the correct amino acids to the correct tRNAs tRNA STRUCTURE Hydrogen bonds between paired bases results in 3-D structure “T-like” structure Amino acids are attached to tRNAs by “aminoacyl-tRNA synthetases” Cells contain 20 different aminoacyl-tRNA synthetases – each recognizes 1 amino acid & its conjugate 1) Start at tRNA site 2) Specific amino acids is bound & activated Translation and Translational Regulation 19:00 3) Specific tRNA bound Translation requires an enzyme to catalyze peptide bond formation Catalyzed by ribosomes (large subunit) Consist of large subunit & small subunit Each subunit is composed of multiple polypeptides & ribosomal RNAs Ribosomes require “help” from proteins to carry out translation 3 STAGES OF TRANSLATION 1. Initiation Ribosome binds at the 5’ UTR & initiates polypeptide synthesis at the start codon...
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2 Membranes are “permeablized” with a detergent(e.g...

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