lab 2 - protein separation

lab 2 - protein separation - Protein Extraction...

Info iconThis preview shows pages 1–2. Sign up to view the full content.

View Full Document Right Arrow Icon
Protein Extraction INTRODUCTION: To separate protein based on molecular weight and to extract a single protein. Initially, we will find a specific protein via affinity chromatography, in addition we will be finding the protein molecular weight via gel chromatography. PRINCIPLE: Three types of chromatography techniques have been observed: Affinity Chromatography, Gel Chromatography, and HPLC. Before choosing a method of chromatography an appropriate buffer must be selected. In chromatography the buffer is important because it is used to hydrate the gel/beads being used. The buffer is used to create a pH range similar to the system that the proteins originated from. By having a pH range around 7.0 the proteins will not denature and we can still work with them. A buffer is better to work, in comparison to water, in that it stays in proximity of a certain pH range. If we were to use water the buffering zone of water is very low, and with any alterations to the system the pH can change thereby denaturing the protein. In addition, a pH meter was used to test the buffering capacity of the solution in order to see if the solution will work best in the gel. Affinity chromatography is protein specific, the proteins are bound via beads that have protein specific ligand attached. This method is being used to separate α -lactalbumin from milk. The separation occurs when the solid matrix attached to antigen, called immunoadsorbent, attracts the α -lactalbumin. After the α -lactalbumin adheres to the beads the remaining protein will flow out of the column. A metalloid reagent is added to the column, the beads thereby release α -lactalbumin. The ligand releases the protein from the beads because the pH of the beads is changed once the metalloid enters the system and the noncovalent interactions are disrupted. After the protein is eluted there are two methods to find out how much protein is in each sample. In order to see protein concentration brown dye is used, brown dye shows the intensity of the proteins that are binding to the paper membrane. Gel exclusion chromatography bears similarities to affinity chromatography, but the major difference is in the function. In gel chromatography the protein is run through the gel and is separated based on the size of the particles. In this protocol we are working with sephadex G-50 beads, these beads fractionation range for proteins is 1,500 to 30,000 Daltons. The proteins are separated due to the beads being porous; the difference beads target a specific range of proteins. Proteins that are too small or too large go through or
Background image of page 1

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

View Full DocumentRight Arrow Icon
Image of page 2
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 5

lab 2 - protein separation - Protein Extraction...

This preview shows document pages 1 - 2. Sign up to view the full document.

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