CaYPD1 purification-1Pre Lab

CaYPD1 purification-1Pre Lab - them to the side of the...

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Pre Lab CaYPD1 purification-1 Purpose: Purification of dry cell pellet of His6-CaYPD1 or CaYPD1-His6. Techniques: 1.Ni-NTA Agarose Beads are agarose beads used for purification of 6xHis-tagged proteins by gravity-flow chromatography. They have nickel-nitrilotriacetic acid (Ni-NTA) resin for proteins containing an affinity tag of six consecutive histidine residues, the 6xHis tag. These beads are used for capturing 6xHis-tagged proteins under native or denaturing conditions. They have high binding affinity and high capacity and are ideal for any scale of purification. Allowing one-step purification of almost any His-tagged protein from any expression system. Assays utilizing Ni-NTA Magnetic Beads involve capture of the 6xHis-tagged protein from a solution followed by washing, binding of interaction partners, further washing, and finally elution of the interacting partner from the 6xHis-tagged protein. Between each step, the beads are collected by attracting
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Unformatted text preview: them to the side of the vessel, after placing near a magnet for 30–60 s. Purification procedures may even use crude cell extracts for binding of 6xHis-tagged protein. 2. Expression systems are used to produce copies of a desired protein within a host cell. T7 RNA polymerase and the lac promoter. Lactose. Steps: Get a column, white lid and yellow bottom lid. Pack a 1mL to 1.5 Ni-NTA Column. Don’t need to use glass rod to pack affinity column. Binding capacity of Ni-NTA is 5 to 10mg proteins per 1 mL resin so 1 column is enough per group. Wash the column with 5 column column dH2O, followed by 5 column volume of buffer 1. Cap the bottom lid, leave 2mL buffer 1 above the top bed of the resin. Label your column with a tape, instead of writing on the column. Cap the top lid tight. Wrap the white cap with Para-film. Then put your column upward in a big beaker and save it in the cold room....
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This note was uploaded on 10/25/2011 for the course ALL 101 taught by Professor All during the Spring '11 term at Cameron University.

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