שיטות להפרדת חלבונים תודה ליורי רזניק ממקיף ח', אשדוד מיכל דייג

שיטות להפרדת חלבונים השפעת ריכוזי מלח וממסים אורגניים. הפרדה על-פי גודל: כרומטוגרפיית סינון בג'ל דיאליזה אלקטרופורזה ב – SDS הפרדה על פי מטען: אלקטרופורזה מחליפי יונים השקעה ב – pI הפרדה על פי זיקה ביולוגית.

Salting out Ammonium sulfate (half saturated) Ethanol (90%) “salting out” adding salt eg ammonium sulfate salting-out effect as [salt]  less H 2 O is available for hydration of protein proteins will aggregate

Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse into the surrounding medium. Dialysis

Gel Filtration Chromatography mix large molecules first

Gel Filtration Chromatography. A mixture of proteins in a small volume is applied to a column filled with porous beads. Because large proteins cannot enter the internal volume of the beads, they emerge sooner than do small ones.

Polyacrylamide Gel Electrophoresis.

Polyacrylamide Gel Electrophoresis. The negatively charged SDS (sodium dodecyl sulfate)-protein complexes migrate in the direction of the anode, at the bottom of the gel. The sieving action of a porous polyacrylamide gel separates proteins according to size, with the smallest moving most rapidly.

SDS-PAGE -S-S- DTT -S-H HS- + -reducing agent -breaks disulphide bonds between subunits Dithiothreitol

SDS - Protein = Sodium dodecyl sulfate SDS-PAGE detergent binds strongly to proteins approx. 1 SDS/2 a.a. Proteins (subunits) become linear Gives all ptns an equal mass : charge ratio

SDS-PAGE

Electrophoretic analysis of serum + - Sample application AnodeCathode + - Separation by charge Albumin  + - Globulins

Ion-Exchange Chromatography. This technique separates proteins mainly according to their net charge. Ion-Exchange Chromatography

Ion Exchange - charge a)Mixture of proteins is applied to the negatively charged b)-ve and neutral proteins pass in the void volume, +ve proteins stick c)Excess of Na + ions is added d)+ve proteins are eluted Could have used change in pH to elute the proteins CATION EXCHANGE

Solubility  fractional precipitation Solubility depends on temperature, pH, ionic strength Increasing temperature generally increases solubility Solubility depends on the pH – minimum at the pI - the isoelectric point – the pH when the net charge on the molecule is zero

Solubility with pH pI is the pH at which the molecules have zero net charge Minimum when pH =pI

Affinity Chromatography. Affinity chromatography of concanavalin A (shown in yellow) on a solid support containing covalently attached glucose residues (G). Could have used change in pH to elute the proteins Affinity Chromatography