BMB 251 1st Edition Lecture 7Outline of Last Lecture I. Purification of a ProteinII. HomogenatesIII. Preparative ultracentrifuge IV. Column chromatographyV. High Performance Liquid Chromatography (HPLP)VI. Affinity chromatographyVII. Fusion proteinVIII. Tandem Affinity Purification-Tapping (Tag-tapping)IX. Sodium dodecyl sulfate (SDS) Gel electrophoresisX. Westerner blotting/immunoblottingOutline of Current Lecture XI. 2-D gel electrophoresisXII. X-ray CrystallographyXIII. Nuclear Magnetic ResonanceXIV. Agarose Gel ElectrophoresisXV. DNA HybridizationXVI. Polymerase Chain ReactionCurrent Lecture- Clicker Question 1: In the gel electrophoresis picture, which amino acid was of the largest size, and which one was the most abundant?o Smaller one amino acids travel farther through the gel, bigger size does not go through very far because they get trapped; ones of the biggest blot (most concentrated area of blue dye) has the greatest number of molecules, so it is of the greatest abundance.- MADLI-TAF: provides an accurate molecular weight for proteins and peptides- Mass spectroscopy: emerged as most powerful method for mapping both post-translational modifications of a given protein- 2-D gel electrophoresis (2 separation procedures): can distinguish up to 2000 proteins by using 2-D protein map (identifies more proteins that SDS gel electrophoresis or ion-exchange can because many proteins have the same intrinsic charges and are similar in size and shape)o Step 1: proteins are separated by intrinsic charges (isoelectric focusing). Every protein has a unique isoelectric point, or the pH at which the protein has no net charge and doesnot migrate into the electric field. Proteins then move to a specific position in the gradient corresponding to their isoelectric point and stay thereThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o Step 2: proteins are put into a second gel electrophoresis containing SDS this time; proteins separate according to size. o **Because this relies on mass spectroscopy, organism must have a completely sequenced genome to use this technique- Chemical biology: inhibitors are detected on living cells, and then used to probe an identity via affinity chromatography of the protein to which it bindso Successfully identified inhibitors of many proteins carrying out key cell biological processes - X-ray crystallography: main technique used to discover 3-D structure of molecules; form of electromagnetic radiation. If narrow beam of x-rays is directed at a sample of pure proteins, most will pass straight through. Yet a small fraction of it is scattered by atoms in the sample diffraction spots are recorded by a detector and analyzed- Nuclear Magnetic Resonance (NMR) Spectroscopy: analyze structure of small molecules o Does not depend on having crystalline sample like x-ray crystallizationo Requires small volume of concentrated protein solution placed on strong magnetic field - ***Because the amino acid sequence determines protein structure and structure dictates biochemical function, proteins that share similar amino acid sequences usually have the same structure and perform similar functions- Agarose gel electrophoresis is used to separate and detects DNA/RNA fragments; similar to SDS gel electrophoresis used with proteins except:o It uses agarose gelo DNA is acidic, so it does not need to be given a negative charge as with denatured proteins because it is already slightly negative from its phosphate group o It uses ethidium bromide staining (fluorescent under UV light)- DNA hybridization is extremely specifico The probe that scientists create is complementary to the specific, desired DNA sequenceand has a radioactive tag attached to ito It will not stick to noncomplementary interactions/sequenceso When it finds the specific sequences, it will create weak interactions (H-bonds) between baseso It then goes through rapid zippering to create the hybrid molecule- Polymerase Chain Reaction (PCR) amplifies a specific DNA fragmento The double stranded DNA is heated almost to boiling in order to break all of the H-bondsand separate the two strandso Primers specifically designed by cell biologists are then add to the DNA strands which then synthesize the new DNA strandso Process is repeated, doubling the amount of DNA with each cycleAs process goes on, more and more DNA are created that are the exact length as specified fragment located between
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