Chapter 3

Chapter 3 - Chapter 3 3.6 Protein Purification Techniques...

Info iconThis preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
Chapter 3 3.6 Protein Purification Techniques Purifications steps usually exploit minor differences in the solubilities, net charges, sizes, and bninding specificities of proteins. Most purification techniques are performed at 0 C to 4 C to minimize temperature dependent processes such as protein degradation and denaturation. First step in protein purification is to prepare a solution of proteins. Isolation of an intracellular protein requires that cells be suspended in a buffer solution and homogenized, or disrupted into cell fragments. Under these conditions, most proteins dissolve. Next step in protein purification is often a relatively crude separation, or fractionation, procedure that makes use of the different solubilities of proteins in salt solutions. Target proteins and other, more soluble proteins remain in the fluid, called the supernatant fraction. Mixture is centrifuged, the fluid removed, and the precipitate dissolved in a minimal volume of buffer solution. Ammonium sulfate gives a two- to- threefold purification. Solvent containing residual ammonium sulfate is exchanged by dialysis for a buffer solution suitable for chromatography. Cylinder of cellophane tubing. Column choromatography can then be used to fractionate the mixture of proteins that remains after ammonium sulfate precipitation and dialysis. As solvent flows through the column, the eluate (the liquid emerging from the bottom of the column) is collected in many fractions. Depends on interactions between matrix and protein. Different proteins are eluted at different rates. The concentration of protein in each fraction can be determined by measuring the absorbance of the eluate at a wavelength of 280 nm.
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Column chromatography may be performed under high pressure using small, tightly packed columns, with solvent flow controlled by a computer. HPLC Ion-exchange chromatography, the matrix carries positive charges or negative charges. Bound proteins can be serially eluted by gradually increasing the salt concentration in the solvent. Reaches a concentration where the salt ions out compete proteins in binding to the matrix. Protein is released and is collected in the eluate. Individual bound proteins are eluted at different salt concentration, and
Background image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 09/08/2008 for the course BCMB 3100 taught by Professor Mendicino during the Spring '07 term at UGA.

Page1 / 6

Chapter 3 - Chapter 3 3.6 Protein Purification Techniques...

This preview shows document pages 1 - 3. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online