11. Pool the fractions containing a high concentration of purified
recombinant proteins, and dialyze extensively against PBS with
two or three buffer changes every 2 h.
3.3 Binding
of Recombinant
Proteins to ECM
1. Protein binding to individual macromolecules of ECM is analyzed in 96-well plates. Prepare vials containing enough
amounts for sampling 1 μg per well. For preparation of laminin,
collagen I, collagen IV, elastin, fibronectin, BSA, and fetuin,
add 5 μL of each component along with PBS to final volume of
500 μL. Dispense 100 μL per well following the example in
Fig. 2 (step 1). The binding assay is performed in triplicate; the
fourth well is used as background. Incubate the plates overnight at 4
C.
2. Plates are washed 3–4 times with the aid of a plate washer. Wash
buffer is used to remove unbound component.
3. Add 200 μL/well of blocking buffer. Incubate for 2 h at 37
C.
4. Wash the plates 3–4 times with wash buffer. When milk is used
as blocking solution, it is important to wash the wells very
carefully to remove milk residue at the bottom.
5. Add 1 μg of recombinant protein in 100 μL of blocking buffer
to each well. Dispense 100 μL/well as shown in Fig. 2 (step 2).
Incubate for 2 h at 37
C.
6. Rinse the plates 3–4 times with wash buffer to remove
unbound protein.
7. Prepare a solution of blocking buffer containing antiserum
against protein to detect the bound recombinant proteins. We
use a dilution where the absorbance equals 1 in titration assays.
Dispense 100 μL/well and incubate for 1 h at 37
C.
8. Confirm the binding using anti-His monoclonal antibodies at
1:10,000 dilution in blocking buffer. Distribute 100 μL/well
and incubate for 1 h at 37
C. As these antibodies are
HRP-conjugated, continue from step 11.
9. Rinse the plates 3–4 times with wash buffer to remove excess
unbound antibodies.
10. Prepare HRP-conjugated anti-mouse IgG diluted to 1/5,000
in blocking buffer, and add the solution to wells as shown in
Fig. 2 (step 3).
11. Rinse the plates 3–4 times with wash buffer to remove excess
unbound antibodies.
12. To develop the reaction, prepare a solution of 1 mg/mL OPD
in 10 mL of citrate phosphate buffer, and add 10 μL of H 2 O 2 ;
dispense 100 μL/well of this reagent. Incubate for 15 min at
room temperature for color development (step 4 in Fig. 2).
13. Add 50 μL/well of 2 M H 2 SO 4 to stop the reaction.
180
Aline F. Teixeira and Ana L. T. O. Nascimento
recombinant proteins, and dialyze extensively against PBS with
two or three buffer changes every 2 h.
3.3 Binding
of Recombinant
Proteins to ECM
1. Protein binding to individual macromolecules of ECM is analyzed in 96-well plates. Prepare vials containing enough
amounts for sampling 1 μg per well. For preparation of laminin,
collagen I, collagen IV, elastin, fibronectin, BSA, and fetuin,
add 5 μL of each component along with PBS to final volume of
500 μL. Dispense 100 μL per well following the example in
Fig. 2 (step 1). The binding assay is performed in triplicate; the
fourth well is used as background. Incubate the plates overnight at 4
C.
2. Plates are washed 3–4 times with the aid of a plate washer. Wash
buffer is used to remove unbound component.
3. Add 200 μL/well of blocking buffer. Incubate for 2 h at 37
C.
4. Wash the plates 3–4 times with wash buffer. When milk is used
as blocking solution, it is important to wash the wells very
carefully to remove milk residue at the bottom.
5. Add 1 μg of recombinant protein in 100 μL of blocking buffer
to each well. Dispense 100 μL/well as shown in Fig. 2 (step 2).
Incubate for 2 h at 37
C.
6. Rinse the plates 3–4 times with wash buffer to remove
unbound protein.
7. Prepare a solution of blocking buffer containing antiserum
against protein to detect the bound recombinant proteins. We
use a dilution where the absorbance equals 1 in titration assays.
Dispense 100 μL/well and incubate for 1 h at 37
C.
8. Confirm the binding using anti-His monoclonal antibodies at
1:10,000 dilution in blocking buffer. Distribute 100 μL/well
and incubate for 1 h at 37
C. As these antibodies are
HRP-conjugated, continue from step 11.
9. Rinse the plates 3–4 times with wash buffer to remove excess
unbound antibodies.
10. Prepare HRP-conjugated anti-mouse IgG diluted to 1/5,000
in blocking buffer, and add the solution to wells as shown in
Fig. 2 (step 3).
11. Rinse the plates 3–4 times with wash buffer to remove excess
unbound antibodies.
12. To develop the reaction, prepare a solution of 1 mg/mL OPD
in 10 mL of citrate phosphate buffer, and add 10 μL of H 2 O 2 ;
dispense 100 μL/well of this reagent. Incubate for 15 min at
room temperature for color development (step 4 in Fig. 2).
13. Add 50 μL/well of 2 M H 2 SO 4 to stop the reaction.
180
Aline F. Teixeira and Ana L. T. O. Nascimento