This article describes how to use protein extraction, quantification, and analysis in the undergraduate teaching laboratory to engage students in inquiry-based, discovery-driven learning. Detailed instructions for obtaining proteins from animal tissues, using BCA assay to quantify the proteins, and data analysis are provided. The experimental procedure requires laboratory equipment and supplies that can be found in most biology teaching labs. Suggestions for successful implementation that can lead to original research published in peer-reviewed journals are outlined.
The benefits of independent research for undergraduate students have been well recognized and documented. In both research and teaching-oriented institutions, research experiences often lead to increased confidence, retention and expansion of knowledge, and career success of undergraduate students across gender and racial lines (Russell et al., 2007). Undeniably, research institutions are able to offer undergraduates more opportunities for scientific discovery in the laboratory than primarily teaching institutions, which have limited time and resources to give high-quality research experience to a large percentage of students (Pladziewicz, 1984; Pierce, 2008).
One solution to the challenges facing educators at primarily teaching institutions is to integrate learning and research in the teaching laboratory. Here, a simple and efficient method for protein extraction and analysis is presented. This method has been successfully used in the master's thesis research of a graduate student at Kean University, as well as in the Independent Research in Biology courses I have been teaching regularly to qualified junior and senior biology majors. Although Kean University is traditionally teaching-oriented and has limited resources for research, I have required all students who took Independent Research under my guidance to perform original, publishable laboratory research to help them develop into future scientists and educators. Understanding how proteins function is essential to the study of biology, as proteins are a major component of the organic materials in any life form and play critical roles in virtually all the cellular processes taking place in any organism. The rapid advances in biotechnology and its increased visibility in our daily lives —— from genetically modified foods, to stem cell research, to genetic testing for hereditary conditions and birth defects —— have enhanced its importance as an integral component of an excellent undergraduate education in biology. It is an enormous challenge, as well as a tremendous opportunity, to engage students from diverse backgrounds in hands-on, inquiry-based, active learning that fosters curiosity and critical-thinking skills that will keep the students abreast with the rapid developments in biology.
By thoroughly preparing the students for the work in advance (e.g., by practicing pipetting, completing calculations for making the stock and working solutions, and understanding the principles of BCA assay), the entire procedure can be completed within a 3-hour laboratory period. This procedure requires the use of laboratory equipment and supplies that can be found in most biology teaching labs, including mortar and pestle, pH meter, incubator, microcentrifuge, hot plate, sterile Epperdorf tubes, dry ice, 96-well flat-bottom microplates, and plate reader. Three chemicals used in the procedure warrant precautions. A fume hood should be used and gloves, lab coats, and safety goggles should be worn when handling PMSF, a toxic protease inhibitor; ββ-mercaptoethanol, which can be absorbed through skin and gives off noxious and combustible vapors; or concentrated HCl, which is corrosive and poisonous.
1. Protein Extraction
Students complete calculations before lab on how much (in grams) of each chemical they need (based on formula weight) to make 50 ml of the following stock solutions: 750 mM Tris-HCl, 1.5 M NaCl, and 10 mM EDTA. Students also complete calculations on how much ββ-mercaptoethanol and PMSF to use (in grams) for making 50 ml of 0.16 M ββ-mercaptoethanol and 1 mM PMSF. At the beginning of the lab, students make stock solutions, adjust the pH of Tris-HCl with concentrated HCl to 8.0, and make 50 ml of the protein extraction buffer (75 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1 mM EDTA, 0.16 M ββ-mercaptoethanol, 1 mM PMSF). The extraction buffer is then stored on ice (Santos et al., 1996). It is important that the extraction buffer be made fresh just before use.
Animal tissues are homogenized quickly with a mortar and pestle over dry ice. The homogenate is then boiled for 3 minutes and centrifuged at 16,000××g for 10 minutes (Yanagisawa et al., 1993). The supernatant is decanted to a sterile Eppendorf tube and stored on ice.
2. BCA Assay
A Pierce BCA (bicinchoninic acid) assay kit is used to measure protein concentrations in the extracts. For animals with an exoskeleton (for example, insects), a 1:25 dilution is recommended. For animals without an exoskeleton, a 1:100 dilution is recommended. In labeled Eppendorf tubes, make the appropriate serial dilutions of the extracts with cold Tris-buffered saline (TBS) and keep them on ice.
Add 10 μμl TBS to wells in columns 1 and 2 (1A to 1G and 2A to 2G) of a 96-well microplate (see Figure 1). Add 10 μμl bovine serum albumin (BSA) stock solution (2 mg//ml) to wells 1H, 2H, 1G, and 2G. Mix the contents of each well with a new pipette tip. Serially dilute BSA protein standard in duplicates from wells 1G to 1B and 2G to 2B. This is done by taking 10 μμl from row G and adding it to row F to mix. Then take 10 μμl from row F, add it to row E and mix, etc. The final 10 μμl taken from row B is discarded.
Pipette 10 μμl of each protein extract (already diluted with TBS) in triplicates into the appropriate wells. Make the BCA working reagent by mixing 10 ml reagent A and 200 μμl reagent B in a 15-ml conical tube. Add 200 μμl of this working reagent to each well used and mix the contents of each well using a new pipette tip. Up to 24 protein samples can be loaded into a single 96-well microplate. With appropriate dilutions of the protein extracts, the sample wells should turn dark green to purple. If the color is too dark, higher dilutions are needed. If the color is too light, use lower dilutions.
Cover the microplate with parafilm and incubate it at 37°°C for 30 minutes. Then remove the parafilm covering and take the microplate to the plate reader to measure absorbance at 595 nm wavelength.
3. Data Analysis
After completing the lab, students are asked to calculate protein concentrations using results of the BCA assay. This is done by constructing a standard curve based on the concentrations of the BSA protein standards and corresponding absorbance values (OD595). To obtain the standard curve, first put concentrations of BSA (from 0 to 2 mg//ml) in column 1 of an Excel worksheet, and put the corresponding average OD595 values from columns 1 and 2 of the microplate reader in column 2 of the same worksheet. Select both columns 1 and 2 of the worksheet, then insert an XY (scatter) chart. From the ““chart menu,”” select ““add trendline,”” then ““linear.”” From ““format trendline,”” select ““set intercept = 0”” and ““display equation on chart.”” This equation displays the relationship between protein concentration (in mg//ml) and absorbance (OD595). From the absorbance values (Y) of each protein extract, the average of all duplicates is taken. The equation is then used in reverse to calculate X. The value of X is the protein concentration of the diluted protein extract. To obtain the concentration (in mg//ml) of the original extract, multiply X by the dilution factor (for example, 25).
Conclusions & Suggestions for Classroom Implementation
The importance of inquiry-based learning, in which the instructors act as facilitators of learning and discovery, has been receiving increased emphasis. Meanwhile, educators face the familiar challenge of having limited laboratory time to present a topic, engage students in hands-on, inquiry-based learning, and find affordable and efficient means for involving them in high-quality, original research.
The procedure for protein extraction and quantification of animal tissues described above has been successfully used with mouse and Drosophila tissues in my Independent Research in Biology courses (Pu et al., 2007). The students and I have done a variety of original research projects utilizing this method. We were able to use Western blot in combination with BCA assay to detect how the expression levels of different isoforms of CaM kinase II, a multifunctional protein present in all animal cells, change in shiverer mutant mice (Suznovich et al., 2007). We cultured wild-type and various mutant strains of the fruit fly, Drosophila melanogaster, under different lighting conditions, and used Western blot along with BCA assay to analyze whether the levels of CaM kinase II are affected by the amount of light the flies were exposed to. In addition to being presented at national conferences, these results have been incorporated into a grant application to be submitted to the National Science Foundation's Research in Undergraduate Institutions (RUI) program. More recently, we cultured wild-type and eye-color mutants of D. melanogaster under various temperatures and measured differences in their protein stability against their reproduction and survivability under each temperature. We employed two methods to measure protein stability, using BCA assay to (1) measure protein concentrations after repetitive freeze-thaw cycles or (2) measure protein decay after a certain amount of time at a particular temperature. The results were presented in a poster at the Annual Meeting of the Society for Developmental Biology in July 2008 and are currently being compiled into a manuscript.
Instructors may wish to break the experimental procedure described above into two lab periods. The first period can be used to explain the experimental objectives and principles of BCA assay and to let the students practice pipetting, make stock solutions, and do calculations for making working solutions of protein extraction buffer. The second period can then be devoted to making protein extraction buffer, obtaining animal tissue, homogenizing to obtain protein extracts, and performing BCA assay. Alternatively, a third lab period can be used by the instructor to help students with data analysis by constructing a standard curve and obtaining the protein concentrations of original protein extracts.
I have found that extraction and quantification of protein is a highly useful technique for students to learn, for a variety of investigative purposes. For example, for measuring protein stability, or in conjunction with Western blotting, this method can be used to compare the expression levels of a particular protein in different tissues or different organisms or after different treatments, or to compare the levels of protein phosphorylation, one of the most important mechanisms to activate or inactivate enzymes and receptor proteins. For instructors interested in adapting this method to plants, algae, or fungi, it would be necessary to modify the components of the protein extraction buffer to take into account the presence of tough cell walls in those organisms. Many different extraction buffers have been used for those organisms, and instructors are strongly advised to consult authoritative Web sites (for example, PubMed) for the recipes to make protein extraction buffer corresponding to the organism of interest.