molecular biology of the cell chapter 8 summary PDF

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Chapter 8: analysing cells, molecules and systems Cell fractionation centrifuging. The goal is to isolate material from eukaryotic cells, like cell organelles. Prokaryotes do not have cell organelles. Large biomolecules like ribosome are DNA can be isolated, for which certain techniques are needed. ...

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Chapter 8: analysing cells, molecules and systems Cell fractionation by centrifuging. The goal is to isolate material from eukaryotic cells, like cell organelles. Prokaryotes do not have cell organelles. Large biomolecules like ribosome are DNA can be isolated, for which certain techniques are needed. o Centrifugation divides cell organelles by a rotor. In a chamber, molecules can be separated. A vacuum is needed for this. The cells and their membranes are disrupted, and the interior of the cell is exposed which is called a cell homogenate. The Cytoskeleton will be at the bottom of the tube, because they have a high density. The other cell elements will still float around in the tube. o Move on to medium speed, so smaller parts of the cell can be separated. Higher speeds means separation of small vesicles or microsomes. o Very high speed is used to isolate ribosomes or virus particles. By applying different speeds, different molecules can be separated. Low speed means big molecules are separated, high speed means small molecules are separated. If you want to isolate protein, low-speed centrifugation should be applied to remove whole cells and other large particles. This is done to study protein structure. Protein purification by column chromatography is where a cell homogenate is applied to a column to purify protein. o Protein can have different binding affinity to the column contents. High matrix affinity stays in the column longer than low matrix affinity. o Add a buffer to the top of the column and the sample will travel through the column. Then collect the sections in tubes, which are called fractions. o Some proteins will travel through the column fast, and some will travel slower due to higher affinity for the column matrix. o If the fractions are collected, the protein ends up in the tube. If the tube is replaced, other proteins are collected in the new tube. o When a lot of tubes are collected, all the tubes have to be checked in order to find the protein of interest. Types of matrixes used for chromatography: A. Ion-exchange chromatography -> Separates the proteins based on charge. Positively charged beads are in the column. When proteins are applied to it, negatively charged proteins will interact tightly with the positively charged beats. Positive or neutral charge will pass quickly through the tube. Salt/sodium chloride is applied to elute the negatively charged protein out of the tube. B. Gel-filtration chromatography -> separates proteins based on size. The columns are made of beads that have small pores. Only small proteins can enter, and large proteins will travel through the column quickly. C. Affinity chromatography -> separates the proteins based on substrate affinity. The beads have a covalently bound ligand. The protein that is able to interact with the ligand is attached to the column material. Proteins that cannot bind the ligand will travel through the column fast. The proteins that are able to interact with the ligand can be eluded through high salt concentration. To discover in which fraction the protein of interest sits, a couple of processes can be done: A. Ion-exchange chromatography -> the amount of collected protein in each fraction is collected. For each fraction, a small amount of protein is taking and added to the substrate. It will be visible where the protein is consumed and the protein which is consumed by the substrate is the protein of interest. B. Gel-filtration chromatography -> after collecting the fractions, add the substrate again. In the fraction of interest, you will find activity. You get more and more of the interest protein, because other proteins are removed. C. Affinity chromatography -> only the proteins that can bind to the ligand can bind the column material. You can elude these by adding a high salt concentration. The activity is measured, and in the fraction with the most activity you have the purest protein. Epitope tagging can be used for the localisation and purification of proteins. Epitope is a molecule that is bound by antibodies. If the gene for the protein of interest is available, add the DNA encoding peptide

epitope tag. A fusion protein is made, making it longer. Put the DNA back into the cell and as a result the cell starts producing the protein, which then fold with the tag attached to it. The tag can be used by purifying the protein with affinity chromatography. A form of epitope tagging is histidine tagging. It is most common, because it very nicely binds to nickel. Nickel is attached to the beads and then the proteins with histidine are applied. The histidine of the protein interacts with the Nickel, they bind to the column material and the protein can be purified. Proteins that not bind the nickel run through the column immediately, which is basically every protein because no proteins have histidine at the end. Different types of protein purification equipment: 1. SDS polyacrylamide-gel electrophoresis (SDS-PAGE) -> is very hydrophobic. Proteins will only run when they are negatively charged. SDS makes all proteins negative charged. The proteins are separated based on size. 2. Isoelectric focusing -> separates protein based on their isoelectric point. This point is that pH where the protein has a neutral charge. 3. Two-dimensional polyacrylamide-gel electrophoresis -> when SDS is combined with isoelectric focusing. You separate on weight and isoelectric point. 4. Western blotting -> used to detect a specific protein out of a whole bunch of protein. The protein is transferred from a gel to a nitrocellulose paper, which is called blotting. The protein is exposed to a specific antibody coupled to an isotype with which you can detect the protein. 5. Mass spectrometry -> used to identify unknown proteins. o Standard mass spectrometry (MS) -> predict the mass of the protein. o Tandem mass spectrometry -> the protein can be sequenced. 6. Fluorescence anisotropy -> measurement of protein-protein binding or protein-ligand binding, based on the tumbling of molecules. When the molecule tumbles fast, it is small. It measures the depolarisation of light. Low anisotropy when the light goes in all kinds of reactions. High anisotropy when the depolarized light will stay in the same form. 7. Phage display -> used to investigate protein-protein interactions in living organisms. 8. Surface plasmon resonance -> used to investigate protein-protein interaction in real time. The change in the angle of incident light is measured in relation to how much protein is added. Beta mercaptoethanol is a reducing agent. It reduces the disulphide bonds that can be formed between two cysteines. Different ways to determine protein structures: 1. X-ray diffraction -> A protein structure can be determined by using X-ray diffraction. For this pure proteins and crystals are needed. Also called X-ray crystallography. 2. NMR -> used to determine protein structure in solution. Conformational changes can be made visible with NMR. DNA nucleotide sequences are recognised by restriction nucleases. Scissors are needed to cut the DNA and glue to put the DNA back together. Restriction nucleases are enzymes that can cleave DNA. There are a couple restriction nucleases: 1. Hae3 -> blunt ends 2. EcoR1 -> staggered ends or sticky ends 3. Hind3 -> staggered ends or sticky ends Most restriction enzymes recognise a sequence in the DNA that has a panaendromic structure. The sequence can be read from left to right or the other way around. It does not make a difference. DNA molecules can be separated using gel electrophoresis. This separates the DNA molecules bases on size. DNA restriction fragments can be easily joined together by DNA ligase, which is a molecular glue that will connect the two ends. It will only work if the ends are compatible. dNTPs fill in staggered ends together with DNA polymerase. The process of ligating molecules together is ATP dependent. DNA can be manipulated to insert a DNA fragment into a bacterial plasmid. A restriction enzyme cleaves the DNA into smaller pieces, with blunt or staggered ends and they can be ligated back together with

DNA ligase. This is an important tool to manipulate DNA and to clone genes. Plasma DNA occur in bacteria. The plasma DNA can be cleaved, the circular DNA will open, and it will turn into a linear piece of DNA. This DNA can be ligated again, with another DNA fragment that needs to be cloned. This results into recombinant DNA. If this circular DNA is obtained, it can be used to determine the nucleotide sequence of the foreign inserted DNA. The synthesis of cDNA from mRNA. Reverse transcriptase is used for this....