with a paper towel. Place membranes in the dish (sample side up).
Pipette ~0.5 mL of alkaline phosphatase conjugated antibody, using
the manufacturer’s recommended dilution (in PBS-Tween, 0.5% skim
milk powder), onto the membrane to form a “liquid pillow.” Incubate
for 1 hour.
Using a torn piece of paper towel, gently soak the conjugate
solution off the face of the membrane and then flood the dish with
deionized water. Agitate for 1 minute.
Transfer the membranes to a new Petri dish containing AP 7.5
buffer (15.75 g Tris-HCl, 5.85g NaCl, 0.4g MgCl2 in 1 L deionized water pH 7.5 with 0.05% [v/v] Triton X-100). Agitate for
Remove membranes to fresh paper towel while you replace AP
7.5 with AP 9.5 buffer (12.1 g Tris base, 5.85 g NaCl, 1 g MgCl2 in
1 L deionized water pH 9.5). Agitate the membranes briefly in the
new buffer and allow to equilibrate for 5 minutes.
Replace with fresh AP 9.5 and continue to wash for another
During the conjugated-antibody incubation step, dissolve 5 mg of
BCIP (5-Bromo-4-chloro-3-indolyl-phosphate) in 100 µL dimethyl-formamide and combine with 10 mg NBT (nitro-blue tetrazolium)
dissolved in 30 mL AP 9.5. This is the color development solution.
Remove the membrane from the dish and blot lightly on paper
towel. Tip AP 9.5 buffer out of the dish and dry with a paper towel.
Place membranes in the dish (sample side up). Pipette ~1 mL of
color development solution onto each membrane.
After 15 minutes, record color development of individual spots.
Samples containing virus turn purple; virus-free samples remain
When color has developed, flood the dish with AP 7.5 and agitate for 2 minutes. Tip off AP 7.5 and flood with deionized water.
Remove and dry the immunoblot (keeps indefinitely).
Samples can be applied to the membrane in other ways. Toothpicks are a cheap and effective way of applying the sap extracts
to the membrane. Sap from herbaceous species such as daffodil
or faba bean can be applied directly by pressing freshly cut stems
to the membrane. Increase the number of field samples applied by
not duplicating samples on the membrane. Students can test 20
samples per membrane and can compare virus incidence between
different sites. This encourages student exploration: they can
compare their lawn or favorite sports ground with other students’
samples. Students then discuss which statistical analyses are suitable for determining whether the differences they observe are
Two or three membranes can be processed in one Petri dish,
thereby reducing plastic and buffer consumption.
A densitometer can be used to measure the intensity of color
development as a proxy for measuring relative virus concentration.
A simpler way to do this is to cover the dish after the color development solution has been applied and score the sample spots as
positive or negative every 2 minutes. Students will get the sense
that the spots develop at different rates. You can then encourage
them to think about and discuss what this means.
Students are required to write lab reports demonstrating their understanding of the immunological/biochemical principles involved in
producing their immunoblot and comparing and contrasting its utility and sensitivity with that of standard ELISA assays (which they
can run in parallel). The learning objective is a deeper understanding
of the strengths and limitations of the different assays. For example,
student-directed research lets them discover that samples can bind
to nitrocellulose under a greater range of conditions than to plastic
surfaces. They are expected to demonstrate their understanding by
generating a graphic similar to Figure 2.
In a third-year biotechnology class, students are asked to regard
the immunoblot as a product they are developing, and they are
assessed on a brochure they produce (see Figure 3) outlining the
benefits of using their kit as well as describing how it works. This
encourages lateral thinking, and some students find it a challenge
not to produce a standard lab report. During the blocking-buffer
incubation step, the students are told they will be interviewing the
technician who set up the day’s lab. They discuss among themselves
and formulate questions they need to ask the technician to gauge the
amount of time and effort required to assemble each component of
the lab. During the conjugated-antibody incubation step, the students interview their technician with the objective of gaining a
deeper understanding of how the lab was set up. They also use this
to develop the sales pitch they will use in their brochure.
Figure 1. An example of a student’s immunoblot.
Figure 2. How the immunoblot works.