14. Read at 492 nm in a microplate reader.
15. Construct the graph subtracting the value of column 4 from
values of columns 1, 2, and 3. Calculate the average and plot
the graph as shown in Fig. 3.
16. ELISA procedure steps including coating, blocking, interaction, and detection are visualized in Fig. 4.
4 Notes
1. For laminin preparation, thaw the solution slowly to avoid gel
formation. It is best to prepare working aliquots.
2. Collagen type 1 can acquire a gel-like appearance when cold; to
solve this problem, warm the solution 37
C. Note if the
product is solubilized.
3. Even after 4 h of dissolving collagen type 4, the solution can
appear hazy with some non-dissolved material. This has been
shown not to affect its biological performance of experiments.
4. Fibronectin should be thawed slowly at 4
C until forming a
clear solution. Avoid vortexing or vigorously shaking the
solution.
5. Different strain can be used as DNA template depending on
the bacterium model used.
6. PMSF has a short half-life; therefore, a freshly prepared solution is used in the purification steps.
Fig. 3 Demonstrative graph of an interaction assay. Absorbance values from the columns 1, 2, and 3 of the
reaction plate are subtracted by values from column 4 (background) of the plate. Absorbance average is
calculated, and then, a column chart is plotted to represent the data
182
Aline F. Teixeira and Ana L. T. O. Nascimento
15. Construct the graph subtracting the value of column 4 from
values of columns 1, 2, and 3. Calculate the average and plot
the graph as shown in Fig. 3.
16. ELISA procedure steps including coating, blocking, interaction, and detection are visualized in Fig. 4.
4 Notes
1. For laminin preparation, thaw the solution slowly to avoid gel
formation. It is best to prepare working aliquots.
2. Collagen type 1 can acquire a gel-like appearance when cold; to
solve this problem, warm the solution 37
C. Note if the
product is solubilized.
3. Even after 4 h of dissolving collagen type 4, the solution can
appear hazy with some non-dissolved material. This has been
shown not to affect its biological performance of experiments.
4. Fibronectin should be thawed slowly at 4
C until forming a
clear solution. Avoid vortexing or vigorously shaking the
solution.
5. Different strain can be used as DNA template depending on
the bacterium model used.
6. PMSF has a short half-life; therefore, a freshly prepared solution is used in the purification steps.
Fig. 3 Demonstrative graph of an interaction assay. Absorbance values from the columns 1, 2, and 3 of the
reaction plate are subtracted by values from column 4 (background) of the plate. Absorbance average is
calculated, and then, a column chart is plotted to represent the data
182
Aline F. Teixeira and Ana L. T. O. Nascimento