BIO 3465–Lab 3-Plant Proteins-Extraction _ estimation

BIO 3465–Lab 3-Plant Proteins-Extraction _ estimation

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BIO 3465 – Plant Physiology Laboratory 3 Extraction, Estimation and Separation of Plant Proteins. Goal: Proteins are major constituents in any organism. Today’s lab will give you an opportunity to learn how to extract proteins from plants and estimate the concentration of total proteins using Bradford method. This will also teach you the basic principles behind protein extraction, spectrophotometry and gel electrophoresis techniques. It will also give a chance to test your own hypothesis on RuBP carboxylase, an abundant protein that is involved in photosynthesis. INTRODUCTION: Proteins are one of the most important components of all living organisms. They are important as structural materials (such as components of plasmamembrane, cell wall, chromatin etc.), or for biochemical or signaling processes (such as enzymes, receptors, ligands, transcriptional factors etc.). When we need to study the function of a particular protein, it is always essential to isolate proteins from plants and then to separate the particular protein from thousands of other proteins. There are many biochemical techniques available today for protein purification. In any protein isolation procedure, the first step is to isolate crude (or total) proteins from plant tissues and then to estimate the protein concentration of the sample. This will be helpful in subsequent procedures either to purify the target protein, or to test expression level of the target protein in a specified tissue or at a specified developmental stage etc. Protein extraction : When extracting proteins extreme care should be taken as protein can be degraded due to protease activity in plant extracts. Generally, proteins are extracted into a buffer solution that contains salts such as NaCl or KCl, protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and/or other commercial inhibitors and a protein stabilizer such as glycerol and surfactants such as Tween or NP40. These various chemicals will help to keep the proteins in their native state as well as to separate proteins from other insoluble materials. When extracting proteins, components of the extraction buffer have to be adjusted depending on your target protein. For example, some of the proteins we discussed during the lecture such as membrane integral proteins and membrane-anchored proteins (that are generally called insoluble proteins) cannot be easily extracted. However, for today’s lab we are only interested in isolating soluble proteins from plants. Why do you think membrane integral proteins are not easily soluble? …………………… ……………………………………………………………………………………………… What type of chemical would you add to the extraction buffer in order to isolate membrane integral proteins? ( think about the membrane protein characteristics that we discussed during the lecture ) ………………………………………………………………………………………………
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This note was uploaded on 11/09/2011 for the course BIO 101 taught by Professor Pesthy during the Fall '07 term at Texas State.

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BIO 3465–Lab 3-Plant Proteins-Extraction _ estimation

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