Techniques In protein Biochem Flashcards
Protein Purifica?on Techniques
Sal1ng Out – separa?ng proteins by their respec?ve solubili?es •
Gel Filtra1on Chromatography – separa?ng proteins by their size • Ion-‐Exchange Chromatography – separa?ng proteins by their charge. • Affinity Chromatography – purifying proteins based on different surface features or ligand binding proper?es. •
High-‐Pressure Liquid Chromatography (HPLC) – high resolu?on method for purifying proteins
Differential Centrifugation as a First Step in Protein Purification
Once the membranes of the cells are disrupted by homogeniza?on or another technique, the organelles and cytoplasmic proteins are released into solu?on. •
Various organelles have different masses that permit them to be separated via differen?al centrifuga?on. •
This technique allows for the isola?on of proteins from specific organelles or the cytoplasm, which can then be further purified.
Salting out
The salt ammonium sulfate, (NH4)2 SO4 , is commonly used to
precipitate proteins. • Proteins precipitate at different concentra?ons of ammonium sulfate, depending on the proteins’ respec?ve solubili?es. •
Centrifuga?on is used to isolate precipitated proteins. •
Dialysis is the process used to remove salt or residual ammonium sulfate associated with the protein.
After centrifuga?on, precipitated proteins are resuspended in buffer and placed inside a dialysis bag. The membrane of the dialysis bag has small pores that allow the ammonium sulfate to diffuse out of the dialysis bag, while larger molecules, such as the protein, are retained in the bag. • •
Gel-‐Filtra?on Chromatography
Separation based on size
The beads that compose the gel filtra?on column have pores of a specific size. •
Larger proteins either cannot enter the beads or spend less ?me in the beads’ pores and thus elute earlier than smaller proteins. • Approximate molecular weight of a protein can be es?mated from a standard consis?ng of proteins of known size.
Small proteins elute less
Ion-‐Exchange Chromatography
Proteins that have a net posi?ve charge (blue) will bind to the nega?vely charged beads (gray) that compose the ion-‐exchange column. •
Posi?vely charged protein bound to nega?vely charged beads are eluted from the column using a gradient of increasing salt concentra?on (example NaCl); salt competes with the protein for binding to the beads. •
Nega?vely charged proteins (red) can be purified on an ion-‐exchange column consis?ng of posi?vely charged beads. •
The charge of the protein is strongly influenced by the pH of the buffer.
Affinity Chromatography
Separa?on Based on Surface Features or Ligand Binding
Small molecules are aaached to beads and a mixture of protein is applied.
Protein that binds the small molecule is retained on the column, while all other protein are washed off the column.
Bound protein can be eluted with a small, compe?ng molecule or a salt gradient. •
Powerful form of chromatography -‐ can yield protein purity >95% in a single step. •
The example above illustrates the affinity purifica?on of a glucose binding protein. •
Examples of other affinity interac?ons: Metal – protein, protein – ligand, protein – protein, enzyme – substrate, enzyme – inhibitor, and an?body – protein interac?ons.
High Pressure Liquid Chromatography
Separation based on polarity
Chromatography is carried out at high pressure, permigng columns with more finely divided beads to be used than in typical columns. • The finer beads provided superior resolving power, allowing high resolu?on separa?on of proteins
Analyzing Proteins by Gel Electrophoresis
PAGE – Polyacrylamide Gel Electrophoresis •
SDS-‐PAGE •
Isoelectric Focusing •
Two Dimensional (2D) Gel Electrophoresis
Polyacrylamide Gel Electrophoresis (PAGE)
Molecules are separated by size and charge in an electric field. • Smaller molecules migrate more rapidly through porous gel matrix. • PAGE separates proteins in their na?ve state (i.e., proteins are folded and any disulfide bonds are intact).
SDS-‐PAGE: Electrophoresis under Denaturing Condi?ons
Sodium dodecyl sulfate (SDS) – a nega?vely charged detergent that denatures proteins. •
Proteins are denatured by SDS and disulfide bonds are reduced by β-‐mercaptoethanol. •
Proteins coated by SDS travel toward the anode (+) at a rate inversely propor?onal to their size (i.e., number of residues) – Shorter proteins travel down the gel faster •
Proteins must be stained to visualize them using a dye, such as Coomassie Blue.
Isoelectric Focusing
Proteins are loaded onto a gel with a gradient of low to high pH values. • A voltage is applied to the gel. • Proteins migrate un?l they reach a pH in which they have a net charge of 0 (defined as the isoelectric point or pI value of the protein).
Two-Dimensional (2D) Gel Electrophoresis
Called 2D gel electrophoresis because the proteins are separated in two direc?ons using different techniques. •
First Step -‐ Separate proteins by isoelectric focusing (separate by charge) •
Second Step -‐ Apply isoelectrically focused proteins to an SDS-‐PAGE gel and separate by their molecular weight •
Individual proteins can then be analyzed by mass spectrometry.
Immunological Techniques Use An?bodies to Purify and Characterize Proteins
Western blogng (protein immunoblogng) • Protein purifica?on by immunoprecipita?on
Antibodies can be made to recognize proteins
The structure of the an?body Immunoglobulin G. The protein’s quaternary structure consists of 4 chains: 2 heavy and 2 light chains linked by disulfide bonds. •
The F ab domain possesses an An1gen Binding Site that binds to a specific an?gen, which could be a protein, carbohydrate, nucleic acid, etc. •
Techniques are available to raise an?bodies against a specific protein.
An?bodies Can Bind Proteins with Very High Affini?es and Specificity
Polyclonal – a mixture of an?bodies that recognize different epitopes (blue, red, and gray) in the an?gen (yellow). Monoclonal – a single type of an?body isolated from an an?body producing cell called a hybridoma
