The use of biotin to demonstrate immunohistochemistry, western blotting, and dot blots in university practical classes
Abstract
Immunological detection of proteins is an essential method to demonstrate to undergraduate biology students, however, is often difficult in resource and time poor student laboratory sessions. This method describes a failsafe method to rapidly and economically demonstrate this technique using biotinylated proteins or biotin itself as targets for detection. This negates the need for a specific primary antibody, saving cost and time. In addition, the easily available and safe reagents allow the methods to be readily adopted without specialist technical expertise. As a result, staff can confidently transfer ownership of the task to the student so as to also develop scientific inquiry skills which promotes student motivation and engagement.
Modern protein chemistry and pathology laboratories commonly use antibodies as detection and diagnostic tools. Therefore, training medical and biological science undergraduates in the practical skills needed for dot blots, Western blotting, and immunohistochemistry is imperative in most courses. Such activities not only support the domain knowledge, for example the selectivity of the antigen–antibody interaction, but they also can be used to develop higher learning that includes important research concepts such as understanding controls and identifying artifacts in results. However, for higher education institutions, running practicals that teach these skills is almost impossible because of the financial burden, time constraints, resource limitations, and obtaining a realistic outcome within the hands of novices [1, 2]. To accommodate students handling small volumes of very expensive reagents in experiments which take longer than a typical 4 hour undergraduate laboratory, sessions often become mainly demonstrations, with key steps illustrated by prepared samples. As a result, the students do not take ownership of the exercise, but instead simply observe and report, and more often than not, this amounts to reporting what another student observed. To overcome this problem, we have developed a series of failsafe, low-budget inquiry-based laboratory exercises that can be tailored to take minimal time, but nevertheless enable the students to gain firsthand experience in the whole process. The methods allow students to undertake crucial steps such as antibody incubations, blocking, and washing with shorter time periods and with no risk of interfering with the quality of the results obtained.
Having a failsafe technique where the outcome is assured meant that the educational process could be freed-up to transfer ownership of the practical to the students. This meant that the students became responsible for some decisions and time management. It also meant that the academics could focus on developing the skills of the students with no impost if the skills were at a low level before they were developed. This report outlines this immunochemistry methodology that is simple and leads to clear student interpretable results. At the same time it develops scientific processing skills in an inquiry-based laboratory task.
METHODS
Overview
The practical involved doing a Western blot, dot blot, and detecting a protein using immunohistochemistry. The activities are suited to being carried out in groups of 2–4 and take 3 × 4 hours to complete. A suggested outline of the activities undertaken in each 4 hour session is shown in Table I.
| Week | Formal instruction | Practical Activity |
|---|---|---|
| 1 | 1. Use of antibodies and their detection | 1. Making 2× SDS-PAGE gels (2.5 hr) |
| 2. Theory of SDS-PAGE, dot blots and immunohistochemistry (1.5 hr) | ||
| 2 | 1. Sample preparation (Western and dot blots, immunohistochemistry) | 1. Decisions on sample loading |
| 2. Controls (0.5 hr) | 2. Loading and running gels | |
| 3. Coomassie staining of gel | ||
| 4. Loading gel and membrane for transfer | ||
| 5. Cutting sections for immunohistochemistry | ||
| 6. Blocking sections (3 hr) | ||
| 3 | 1. Interpretation of results (0.5 hr) | 1. Developing western blot |
| 2. Carrying out dot blots | ||
| 3. Developing immunohistochemistry sections and microscopic analysis (3.5 hr) |
In overview, for the Western and dot blots, the positive control sample, bovine serum albumin (BSA), and a mixed protein sample (BS, bovine serum) were spiked with biotinylated BSA (B-BSA). This meant that no antibodies were needed in the reactions but instead a buffer or water could be used as a surrogate. For the penultimate incubations, an avidin-linked enzyme was used to bind to the biotinylated protein before the color was developed. For the immunohistochemistry of the practical, it was found when perfusing the vascular system of a rat with a biotin solution for another purpose, the biotin specifically bound to the endothelial cells in the kidney. Interestingly biotin also binds to heart endothelial cells [3], but this was not examined here. Afterwards, the tissue was fixed, embedded in paraffin, and sections prepared. To be consistent with the eventual staining pattern obtained, the students were told that they would be using a primary antibody for detecting kidney endothelial cells. The sections were then processed in a similar manner to the Western and dot blots, that is with no need for primary or secondary antibodies.
Student Profile and Background
The majority of students (52%) who attend the University of Western Sydney (UWS) are the first in their family to attend university and around one-third (36%) come from a non-English speaking background. Immunology is a Level 3 unit taken in the first semester of the final year by undergraduates in life science degrees (Bachelor of Science, B.Sc./Teach or Bachelor of Medical Science degrees) which require the completion of 24 units. Level 2 biochemistry is a prerequisite and almost all students have done a microbiology unit. The students had little prior experience of inquiry-based learning and little confidence in decision making, but all were competent in basic laboratory skills such as pipetting and maintaining a laboratory notebook.
Session 1
The main practical outcome for Session 1 was for each group to have prepared 2× 10% Tris-HCl SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels with 6% stacking gels containing lanes for protein application [4]. One gel was to be used for detection of total protein and the other for Western blotting in the next session. Practical procedure handouts (see Supporting Information material) were not protocols but broad instructions highlighting what needed to be done. For the assemblage and running of the gel apparatuses, students followed manufacturer protocols, which gave them exposure to professional-level literature.
Educational instruction centered on giving an overview to the practicals, which included: a review of protein separation by SDS-PAGE; basic principles of immunolabeling; and a discussion on the similarities of and differences between the techniques to be used. The concept of gel electrophoresis for protein size determination was reviewed. No student had previously carried out the technique, but all had basic knowledge in protein structure and function. Sizes of proteins were discussed as well as the dynamic range of size separation with differing acrylamide concentrations in the SDS-PAGE gels. Other issues addressed included sample preparation issues (e.g. soluble versus non-soluble proteins, native versus reducing gels, detection limits of stains, protein membrane transfer, and colorimetric antibody localization methods). The expected result of dot blots without the dimension of protein separation and therefore molecular mass determination was also discussed. The groups were then asked to design their experiments to: (a) identify the molecular mass of BSA in a sample of BS; and (b) to estimate the quantity of BSA in BS. This was followed by a whole class session, where the groups discussed their ideas and were guided into a common protocol by the lecturer. Particularly important here was to develop a broad discussion about the need for positive and negative controls as well as non-specific binding and quantification limits. It was also used to ensure that all groups had a good understanding of the practical techniques that would be followed.
Session 2
The main practical outcomes for Session 2 were for each group to have run and stained an SDS-PAGE gel, to have transferred the proteins of their second SDS-PAGE gel to a membrane ready for Western blotting, and to cut wax sections for immunohistochemistry. The materials required for the Western and dot blots were: horse serum (HS) and BS from Oxoid (Hampshire, UK), BSA; biotinylated-BSA1 (B-BSA), ExtrAvidin-alkaline phosphatase and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium SIGMA-FAST tablets from Sigma (St. Louis, MO, USA), biotin, nitrocellulose, and broad range Kaleidoscope markers from BioRad (Hercules, CA, USA). Some of the stock solutions given to the students were spiked with B-BSA, but they were labeled as if they were without the B-BSA (Table II).
| Solution | Label |
|---|---|
| BS with 350 μg/mL B-BSA | “BS” |
| BSA 0.5 mg/mL | “BSA (0.5 mg/mL)” |
| B-BSA 350 μg/mL | “BSA (100 ug/mL) for Western Blots only” |
| B-BSA 350 μg/mL | “BSA (40 mg/mL) for dot blots only” |
| BS | “HS” |
| BSA (0.5 mg/mL) | “HSA (0.5 mg/mL)” |
- Solutions indicate components to be made up by technical staff however are labeled as indicated for student use.
- BS: Bovine serum, HS: Horse serum, BSA: Bovine serum albumin, B-BSA: biotinylated bovine serum albumin, HSA: horse serum albumin.
Proteins were separated on the SDS-PAGE gels using Tris-glycine buffer (25 mM Tris, 0.192 M glycine 0.1% w/v SDS). Samples were boiled in sample buffer (63 mM Tris pH 6.8, 25 % (v/v) glycerol, 2% (w/v) SDS, and 0.01% (w/v) bromophenol blue, 350 mM DTT) for 5 min before loading 0.5–2 μL of serum or 20–500 ng of BSA onto the gel. The kaleidoscope molecular mass markers were put into a lane designated by the group. The amounts of protein were determined by the groups and there were enough lanes to do more than one amount. The gels were run at 200 V for 1 hour. The gels were transferred either into Commassie Blue stain (0.1% (w/v) Commassie Blue in 40% (v/v) methanol, and 10% (v/v) acetic acid for 30 min or into transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) for 20 min on a rocking table. Commassie stained gels were destained in 40% (v/v) methanol and 10% (v/v) acetic acid. Technical staff completed the destaining process after the session and when background staining was reduced, gels were photographed. The duplicate gel was used for Western blotting. Proteins were transferred to nitrocellulose using a Mini Trans-Blot electrophoretic transfer cell (BioRad) with the students using the manufacturer's instructions. The cell was run at 100 V for 1 hour at 5°C. Membranes were removed from the apparatus, and transferred into phosphate buffered saline (PBS) and stored at 5°C. To demonstrate to the students whether or not the protein transfer had taken place, this duplicate gel was also stained with Coomassie Blue (see above) after the protein transfer.
Approval for the use of animals in teaching was obtained from the relevant Animal Care and Ethics Committee. To prepare the tissue for immunohistochemistry, a rat was anaesthetized with Fluothane (other general anesthetics could also be used), the chest opened, and 0.5 mL of heparin was injected into the left ventricle. The animal was then perfused through the left ventricle with ∼150 mL PBS at room temperature at a pressure of about 110 mmHg (5–7 min). Just before the end of the saline perfusion 20 mg of Biotin was dissolved in 30 mL PBS. It was important to make-up the Biotin solution just before use. The perfusion was then switched to the Biotin solution, and then back to the PBS to wash with about 100 mL of PBS. The kidneys were removed and immersion fixed in 4% phosphate buffered paraformaldehyde for 12 hours before processing for wax sections. The kidneys were rinsed in PBS, dehydrated through alcohols, cleared in xylenes, and embedded in wax. One of the wax blocks was trimmed and prepared for sectioning in the microtome by the instructor and students were shown how to section and float out sections onto glass slides. Each student was able to quickly cut a few sections and make one or two of their own slides which were set aside for the next session.
Educational instruction centered on giving an overview of the Western blotting procedure, including the transfer of proteins to a nitrocellulose membrane and discussions were held on the amount of protein to be loaded onto the gels, and the purpose of the molecular mass ladder. The instructor demonstrated loading of a protein sample into one of the wells of the gel, after which the students were alone to load their own samples. Once the gels were running, the principles of immunohistochemistry were explained and the students were asked to compare this process with the Western blots. The methodology for fixing and embedding the tissue in wax was described and the possible effects of fixation, dehydration, and embedding on antigenicity were discussed. Once the gels were removed for staining or transfer and while the transfer process was occurring, the groups were shown how to cut sections and the students prepared one or two slides of their own.
Session 3
The main practical outcome was to immunostain the Western blots, prepare and stain dot blots, and carry out the immunohistochemistry on the sections. Essentially, these processes were carried out in parallel so that the students could appreciate the similarity in the techniques.
Western Blots
Membranes were transferred into the blocking agent, 0.5 % (w/v) skim milk in Tris buffered saline (TBS; 20 mL), the night before class. Students were asked to place 1 μL of H2O (labeled: mouse monoclonal anti-BSA “primary antibody”) and the membrane incubated for 10 min at room temperature on a shaking platform. The “primary antibody” was drained off and the membrane was washed in TBS 2× 5 min. The “secondary antibody”, ExtrAvidin-alkaline phosphatase was then added at a 1:20,000 (v/v) dilution (labeled: alkaline phosphatase conjugated goat anti-mouse IgG ”secondary antibody”) in TBS and incubated for 1 hour on a shaking table. The “secondary antibody” was drained off and the membrane was washed in TBS for 2× 5 min. Detection was using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium SIGMA-FAST tablets and the reaction stopped by removal of substrate and washing the membrane with water.
Dot Blots
On a nitrocellulose membrane, a grid was marked out with a soft graphite pencil. The membrane was then wetted with MeOH and partially air dried before placing dilutions of the samples of spiked BSA (labeled “BSA 40 mg/mL”), spiked BS (labeled BS), and HS. Students were told to determine their own loading of samples, but typically used 2- to 10-fold dilutions of the serum samples and control samples which resulted in 10–100 ng of protein being applied to the nitrocellulose. Dot blots were then blocked in 0.5% (w/v) skim milk for 30 min and then processed as above for the Western blot.
Immunohistochemistry
Prepared sections were then given to the students, one labeled test and the other labeled control. The test section was from the biotin perfused animal and the control section was from a kidney of an animal that had been perfused and fixed without prior treatment with biotin. The control sections had been pre-prepared by technical staff. The students then organized in their group for one of them to dewax and hydrate the sections. Sections were then allowed to air dry. The sections were then sequentially incubated in PBS containing 5% serum for blocking, primary antibody which was PBS with 5% serum (labeled: sheep anti-endothelial cell antibody “primary antibody kidney”), washed in PBS-serum 2× 5 min, incubated with ExtrAvidin-conjugated horseradish peroxidase (Sigma, Sydney, Australia) (labeled: peroxidase conjugated donkey anti-sheep IgG “secondary antibody kidney”) for 30 min, washed 2× 5 min and then incubated with a solution prepared from a diaminobenzidine substrate tablet in the presence of hydrogen peroxide. Students observed microscopically the wet preparation under low power to monitor the color development, and once adequate color had developed, the sections were washed with TBS and placed in a slide rack in water. The control sections were processed in the same manner, but with pre immune serum replacing the primary antibody. The pre immune serum was PBS with 5% serum (labeled “pre-immune sheep serum”). The technical officer then batch processed the slides counterstaining them with haematoxylin, dehydrating, clearing, and mounting the sections under a coverglass. This was done in class so that the students could observe the process.
RESULTS
Session 1
Students were provided with stock solutions that they had to dilute to working concentrations. This took considerable time and discussion as most students had little confidence with their calculations. The students were allowed to proceed on the basis of their calculations and to set up the gel pouring apparatus using the professional instructions. This created a collegial, if not social atmosphere between the groups as they worked together to determine how to pour the gels. The supervisors gave minimal input to this process, but did instruct groups to pre-test the integrity of gel assembly apparatuses for leakage using 70% ethanol prior to pouring acrylamide into the gel casting apparatus. In some cases, polymerization did not occur in either the main gel or the stacking gel, mainly due to incorrect calculations or mixing, and leaking still occurred in some cases. The design of the practical class left ample opportunity for students to repeat the process if they were unhappy with the quality of their gels. It was common for groups that had to repour their gels to seek advice from other groups and the instructor. In the rare instance where students were left with no gels, staff prepared “spare” gels before the next session. After polymerization of the separating and stacking gels, students critiqued the overall quality of their own gels and examined why some gels did not work. The groups were given homework to decide on the preparation of samples for loading before the second session.
Session 2
Students were instructed to start loading and running their gels based on decisions made outside of class. The time lag between entering the classroom and loading of gels was dependent on whether the groups had discussed the intended loaded samples before coming to class. Students were expected to approach staff when they had group consensus on volumes of samples and controls for the experiment. After instructor clearance, students loaded and ran gels according to the manufacturer's instructions. No instructions were given to the students about recording of group decisions including the order of loading samples and molecular weight marker ladders onto the gels. Once the gels were run, minimum instruction was given to the students in how to remove the gels and handle them, but the 10% polyacrylamide gels were relatively robust under student handling. A few gels broke at this stage, but this gave the opportunity to show students how to piece them back together again so that the information was not lost.
Figure 1 a shows a typical student generated SDS-PAGE gel stained with Commassie Blue, a result usually generated in the last 30 min of practical class in Session 2. The separation quality of these gels varied, but often exceeded those of the commercial gels used by some students in the laboratory (results not shown). Students placed different amounts of samples on the gels, while others ran duplicate samples (as in the illustrated gel). It was stressed that these sorts of decisions were made daily at the bench depending upon the research question. The complexity of protein components in serum was discussed and often for the first time students visualized a complex protein sample such as serum. The use of authentic standards indicated to the student where on the gel the albumin may be migrating, however whether it was present or not, students clearly decided that this could not be determined with only PAGE as evidence. Discussion about the relative intensity of staining of proteins was also introduced at this point.
(a) Student generated Commassie stained 10% SDS-PAGE gel of 1 μL of BS containing 350 μg/mL biotinylated BSA (Lanes 1 and 2), 100 μg of BSA (Lanes 3 and 4), 1 μL of BS labeled “HS” (lanes 5 and 6) and 100 μg of BSA labeled “HSA” (Lanes 7 and 8). (b) Western blot analysis for detection of biotinylated BSA using alkaline phosphatase conjugated extra-avidin. 1 μL of BS containing 350 μg/mL biotinylated BSA (Lanes 1 and 2), 350 ng of biotinylated BSA (labeled BSA) (Lanes 3 and 4) 1 μL of BS labeled “HS” (Lanes 5 and 6), 350 ng BSA labeled “HSA” (Lanes 7 and 8).
The packing of the gel with supports and blotting paper for transfer to the nitrocellulose membrane was demonstrated briefly to the whole class and then the students were left to their own devices. Discussions were held about the heat generated during wet transfer of proteins from gels to membranes and why it was important to remove the heat. The prestained protein markers being indicated on the membrane and the lack of Commassie stained proteins in the transferred gel also gave students an indication as to the success of the process at the end of Session 2.
For histochemistry, most students were a little awkward handling the sections but greatly appreciated learning about the processes that gave rise to the microscope images they had seen through out their previous studies. Many were unaware of the processing and staining of histological slides. The opportunity was taken to discuss occupational, health and safety issues associated with the process—exposed sharp knives and handling xylenes.
Session 3
The use of biotinylated proteins in the specimens allowed the process to be shortened with truncated time periods for mock exposure to primary antibodies. Diluting stocks of “mock primary antibodies” gave the students most concern as they were not confident with calculations. Having “mock primary antibodies” meant that we could get them to redo their dilutions if necessary, or were unconcerned if they used a large amount of “mock primary antibody” to make their dilutions. The instructor took the opportunity to discuss expense and waste with the groups if this happened. Membranes still required blocking with skim milk and subsequent washing of the membrane before the addition of mock conjugated secondary antibodies (ExtrAvidin labeled alkaline phosphatase). As seen in Fig. 1 b, BS (spiked with B-BSA) and the positive control of BSA (spiked with B- BSA) show a strong signal at the molecular mass expected (∼67 kDa) and other bands at higher masses were discussed and explained as aggregates. The authentic standard (Fig. 1 b, Lane 3) confirmed the identity of the band. During this time, discussions centered on the negative controls (HS, Fig. 1, Lanes 6 and 7) and other appropriate controls that could have been undertaken (e.g. the use of pre-immune serum instead of primary antibody, the addition of free BSA in the incubation with the membrane and primary antibodies).
The opportunity in Session 3 to rehandle membranes in carrying out dot blots ensured students were primed with respect to the use of gloves, forceps, and fragility of material. Students were confident in both wetting the membrane (a task repeated from Session 2) and applying the protein. The repeating of the processing of the membrane, being identical to that of the Western blot, clearly gave students the confidence to undertake this activity independently. Figure 2 shows the varied quality of dot blots produced in the laboratory by students. Students decided on controls and dilutions of the standards provided. Some students even made samples of HS and BS with BSA added to assess any blocking of the signal in the background of other proteins present.
Student generated dot blots of various qualities for the detection and quantification of biotinylated BSA in bovine serum. (a1): 2 μL of “BS”; (a2): 2 μL “BS” diluted 1 in 2; (a3): 2 μL of “BS” diluted 1 in 10; (a4): 2 μL “BS” diluted 1 in 50. (b1): 2 μL of “BSA (40 mg/mL) for dot blots only”; (b2): “BSA (40 mg/mL) for dot blots only” 2 μL of 1 to 1 dilution; (b3): “BSA (40 mg/mL) for dot blots only” 1 in 10 dilution; (b4); BSA (40 mg/mL) for dot blots only 1 in 100 dilution. (c1): 2 μL of “HS”; (c2): 2 μL of “HSA”; (c3): 2 μL of 1 in 5 dilution of “HSA.”
Immunohistochemistry staining of sections was observed in real time using microscopy while product was developing. Students were confident in light microscopy, but the correct method for Kolher illumination needed to be reinforced. A typical section obtained with this technique is shown in Fig. 3. The glomeruli were clearly stained and the tubules were not. There was great excitement to see the outcomes, and the difference between identifying that a kidney had the antigen of interest (Western blot technique) versus identifying the location of the antigen in the tissue (immunohistochemistry) was discussed. Again controls were considered with the various combinations of pre-immune serum and staining sections with and without primary or secondary antibody. Possible problems caused by the presence of endogenous biotin in the tissue were also discussed in relation to the appropriate controls that could be used to determine this.
Pseudo-immunohistochemical result of kidney for the location of biotin after biotin perfusion. Note that the endothelial cells of kidney blood vessels stain and the tubular epithelial cells are unstained. Scale = 100 μm.
Student Outcomes
The students were informally observed by teaching staff for engagement and confidence in decision making during the three sessions. Staff noted that the independence of students grew during the three weeks as indicated by the students being more confident in their own skills and decision making abilities. Most student anxiety centered on calculations, a skill scaffolded from year 1. Students struggled with the concept of amounts versus concentrations. For some students the realization of the importance of the skill became clear given that a lack of competency would have consequences for their results. Students were often heard to comment that “calculations were taught in chemistry or biochemistry” indicating that they were starting to decompartmentalize their learning. In contrast, students were observed to confidently interpret and use professional level literature and in the most part were able to adapt these to their own needs.
The students had clear goals to reach at the end of each week, something they could confidently critique with minimal instructor feedback. For instance, they were able to decide on the quality of their poured gels, Commassie Blue stained gels, and finally their dot and Western blots. The majority of students were engaged and attempted to solve problems in groups and took responsibilities for their actions. This often resulted in the group deciding whether to repeat a task. When students did approach staff, their inquiries were pro-active, for example they indicated that had misinterpreted instructions or had issues with skills such as gel loading. This meant that staff were involved in trouble shooting rather than giving detailed instructions prior to students attempting the task. Staff also noted the students moving from group to group to assess results of different gels/blots; something that was not usually observed in other biological sciences classes. This may have been because the outcome of the practical was visual with no need to manipulate data or represent data in graphs to assess the success of the activity.
Student record keeping using a laboratory note book needed supporting by staff, despite the fact that this skill was scaffolded from second year. As there were no formalized laboratory methods provided, the group decisions needed accurate recording as did the results and their interpretation. Students still had the tendency to write things on scraps of paper (to be “written up later so my lab book will be neat”), and not record precise details like units or even the loading order of gels. To be able to record scientific activities with accuracy was also supported in other units that the students were doing concurrently with immunology.
“More help in lab”,
“Better to have methods or procedures in lab manual”.
and “Practicals needed more explaining”.
“practicals (simple and fast)”
“practicals that covered techniques that I really enjoyed.
“practical section for exposure of different techniques.”
“lab sessions, because we experienced carrying out procedures that we had only previously read about e.g. western blots and ELISAs”
“labs [that] taught a range of skills”
“lab classes [that] were conducive to learning, allowed for more practice of skills”
From the comments above, students recognized that the outcomes of the practicals were skill development and task design rather than a stained gel or histological section. The students also saw the skills as different from other biological science techniques they had been exposed to.
DISCUSSION
This inquiry-based practical was designed to expose students to some of the techniques central to immunology and used extensively in biological sciences laboratories. The importance of these techniques is reflected in the vast array of literature reporting method development over the last 30 years [5-7]. Inquiry-based learning is when students are asked to design and complete experiments where they develop their learning in scientific thinking [8]. Having students self-assess and repeating components of practical activities are also indicators of inquiry-based activities [9]. These are important components of life-long learning processes for science graduates where inquiry asks students to show understanding of scientific process. The benefits for students also include deeper understanding of content, positive confidence, and attitude to performing science and higher retention rates in courses [10].
As pointed out by others, often laboratory experiences are busy and confusing for students as they grapple with new theories and language around methodology, new equipment, decision making and recording and analyzing results [11]. For this reason, the science behind the constituents of sera was not directly taught in this class, but was used as vehicle to achieve the main outcomes from this activity, which were the acquisition of skills in the techniques and the development of confidence in decision making. The overlay of the different practicals also enabled students to recognize how good time management through planning of activities allowed a large amount of work to be achieved without a lot more effort. This required the supervisory staff to remind students at various times at which stage they should be and what they needed to be planning or doing next.
The use of professional literature for decision making, albeit with some generic written material to direct students, legitimized the difficulty of the task to that expected of a graduate. It also made the task authentic whether the student envisaged a future in research, industry, or non-laboratory based careers. The direct demonstration that students can interpret and use this level of material reinforced student confidence as they acquire graduate attributes such as being able to “manage multiple information sources and new and complex challenges” [12, 13]. At this stage of their education, the movement away from a “cook book” laboratory task was possible with the benefits including increased student engagement and allowing students to consider the purpose, order, and time management of tasks [14]. Analogous to other studies where students have had little exposure to inquiry-based learning, there were still elements in the cohort who struggled to adapt to this approach [15]. The feedback from these students indicated that they failed to realize that experimental design and decision making was just as an important outcome as the quality of results obtained.
Besides the obvious advantage of this technique in saving time and cost, the activity is a pedagogical niche between what amounts to a full research activity and a “cook book” experience typical of using commercial kits. Many immunological practicals described in the literature focus on science content rather than the acquisition of skills [16, 17] and hence tend towards the unpredictability of a full research experience. In addition, they often require very specialized techniques and expertise to prepare the activity, for instance, fruit fly colonies or specialized cell culture techniques [16, 17]. The other extreme is the use of kits where decision making and preparation is removed from the activity. Our activity reduces the risk of a lack of outcome from the activity which frees up staff to ensure development of skills and inquiry-based learning without dividing the emphasis of the activity between the science and the skill acquisition.
Although a specific outcome must occur in the practical, it should be stressed that this was not in the context of the results becoming predictable; much of the decision making was left up to the student which meant they were involved in the design of the task and this in turn increased their motivation and interest in outcomes [18]. Formal instruction, especially in the first session, made sure that students had the prerequisite background so to give the opportunity for early entry into the task and allowed maximum involvement. The visual nature of the experimental outcomes ensured peer review and prompted wide discussion throughout the class. This self-reflection and the decision to repeat and/or trouble shoot tasks was indicative of students being motivated and engaged. In our case, we chose not to reveal that this practical was a sham, but depending upon the pedagogical focus, this could be stated at the outset. In particular, such disclosure would be useful, if this were part of a more extensive program leading to development of research skills. In our case the purpose of the activity was not to give the students a research experience, but to learn and gain confidence in these fundamental skills.
The three elements of the practical (Western blotting, dot blotting, and immunohistochemistry) can be adopted in their entirety or be taught independently depending upon resources available. For instance, if the technical expertise for the initial preparation for the sectioning of tissues is not available, then this could be deleted from the practical. Otherwise, the procedures outlined in this article use reagents commonly available in biological laboratories. The exception was the biotinylated “target protein,” which could be purchased or prepared by technical staff if a specific biotinylated protein were required.
In conclusion, this easy and safe laboratory activity can be implemented in biological science undergraduate laboratories with a limited expertise. The optimized method also tested rabbit, horse, and sheep sera instead of bovine serum as the background material for spiking with the biotinylated protein. These showed similar results with around 34 μg total protein showing optimum staining by Coomassie blue after electrophoresis. The nature of the target protein also can be altered as desired by staff to illustrate a range of inquiry-based activities based on the availability of biotinylated proteins or tissues to allow student ownership and development of science inquiry skills. However, like a range of other student centered activities, the resistance of students to these types of innovated teaching requires staff to constantly reinforce the intended outcomes of the activities. This process may also need to be scaffolded throughout programs so students get consistent messages as they progress from subject to subject during their degree.
