Upon completion of this module topic, you should:
- Be able to extract and isolate proteins from a tissue or cell sample.
- Be able to purify proteins using precipitation (immunoprecipitation) techniques to isolate specific proteins from a sample and to study protein-protein interactions.
- Be able to conduct absorbance (spectrophotometry) and colorimetric (Bradford) protein assays to measure and quantitate protein concentrations in a sample.
Protein Extraction, Precipitation, & Separation
This is Part a, Protein Extraction, Precipitation, & Separation, under the module topic Protein Techniques. This topic part has three sections to review: Content Tutorial, Animations & Activities.
An Introduction to Protein Purification
1. An Introduction to Protein Purification: University of Calgary Biotechnology Training Centre
- Protein Purification Introduction PowerPoint (heart.jabsom.hawaii.edu, Powerpoint Document) Powerpoint: – General Considerations – Expression Systems
2. Protein Purification Introduction: MIT Open Courseware
- Protein Purification Introduction (ocw.mit.edu, Multimedia Page)
*Note: It is recommended to visit the Qiagen website listed below. This website is integrated into the MIT Introduction and it provides Protocol Images.
- Qiagen Website (www1.qiagen.com, PDF Document)
3. General overview of techniques for separating and purifying proteins:
National Taiwan University- Department of Biochemical Science & Technology
- Basic Protein Purification (heart.jabsom.hawaii.edu, Powerpoint Document) (Reference: R.H. Juang (2004) BCbasics)
4. Introduction to Protein Purification
- PowerPoint (heart.jabsom.hawaii.edu, Multimedia Page)
Purification Scheme – Protein Fusion / Purification – Overview of Standard Methods
1. Simple and Reliable Method to Precipitate Proteins from Bacterial Culture Supernatant, Caldwell, R.B., and Lattemann, C.T. (Applied and Environmental Microbiology, 2004, pp.610-612)
- Article (heart.jabsom.hawaii.edu, PDF Document)
1. Ion-Exchange Chromatography Animation: Wiley Science
- Ion Exchange Chromatography (www.wiley.com, Multimedia Page)
2. Gel Filtration Chromatography Animation: Wiley Science
- Gel Filtration Chromatography (www.wiley.com, Multimedia Page)
3. Co-Immunoprecipitation to Study Protein-Protein Interactions (Promega – TNT System)
- Protein Interactions (www.promega.com, Multimedia Page)
This is Part b, Protein Quantitation, under the module topic Protein Techniques. This topic part has two sections to review: Content Tutorial & Animations.
2. Rice University Experimental Biology
Click on the following link to view a tutorial an introductory tutorial about methods for setting up protein assays and measuring protein concentrations through spectrophotometry. The tutorial consists of three webpages that you can navigate through using the purple arrow on the right side of each page.
The tutorial is organized into the following three sections:
- Colorimetric Assays
- Principles of Spectrophotometry
- Protein Assays
- Protein Assay Tutorial (www.ruf.rice.edu, Multimedia Page)
3. Bio-Rad Laboratories
The following link will direct you to a pdf document of the Bio-Rad Laboratories Protein Assay Manual. The Bio-Rad protein assay is analogous to the Bradford Assay. The manual contains an introduction to, a description of and common questions about the technique.
- Bio-Rad Protein Assay Manual (labs.fhcrc.org, PDF Document)
Biuret Protein Assay Activity: Reference- Western Kansas University, Biology In this activity your will conduct a virtual Biuret protein assay to measure the concentration of protein in samples that are of known values (standards) and of unknown values. The spectrophotometer will be used to measure the absorbance of each sample. In the first part of the experiment, you will prepare a standard curve that will plot your standard absorbance values as a function of protein concentration. As you use the virtual spectrophotometer to measure the samples, you will record the absorbance value in the data table provided and then you can prepare your standard curve. There are two follow-up questions that you can complete before moving on to the second part of the experiment. In the second part of the experiment you will be provided with two unknown samples in which you will measure the absorbance. The concentrations of the unknown samples will be compared to the known standard samples that you have plotted on your standard curve. There are five follow-up questions to the second part of the experiment. *Note, you do not need to enter your name at the conclusion of the lab.
- Protein Assay Virtual Activity (bioweb.wku.edu, Multimedia Page)