173
supernatant into a syringe and attach a 0.45 μm filter to the
end. Filter the supernatant to remove large particles. Replace
the filter if it becomes clogged.
3. Equilibrate a 1 mL Nickel Sepharose His Trap column with
Lysis and Binding buffer by washing the column with 10 column volumes. Load the clarified protein lysate through the
column. Keep an aliquot of the lysate and flow-through for
analysis. Wash the column with 10 column volumes of Lysis
and Binding buffer and collect an aliquot for analysis. Elute
with 10 mL of Elution Buffer. Load equal volume fractions of
all collected aliquots on a SDS-PAGE gel to estimate the efficiency of purification. Add glycerol to a final concentration of
50% and store at −20 °C.
4. Alternatively, if using an FPLC instrument, equilibrate column
with 10 column volumes of Lysis and Binding buffer. Clarified
lysate is loaded into the system at a flow rate of 1 mL/min.
Wash column with 10 column volumes of Lysis and Binding
buffer. Samples are eluted using gradient length of 25. The
entire run can be done at a flow rate of 1 mL/min with the
pressure kept below 0.30 MPa.
1. The assays are generally carried out at room temperature in a
384-well transparent bottom plate.
2. The exact concentration of the sensor and protease or macromolecular binder must be determined empirically. Before starting the assay, test a range of sensor concentrations with varying
concentrations of protease or macromolecular binder to determine the point of optimal sensitivity.
3. When setting up multiple reactions, it is desirable to make a
master mix containing the common components. The typical
reaction in a total volume of 25 μL is set up as follows.
4. Add a suitable volume of PBS that will eventually make up a
total reaction volume of 25 μL (i.e., we add PBS first to prevent small volumes of other reagents from sticking to the side
of the well or remaining stuck to the pipette tip).
5. Add 2 μL of diluted sensor to a final concentration of approximately 5 nM and mix with 2–5 μL of various dilutions of protease
or macromolecular binder. The final optimal concentration of
components needs to be determined empirically. For enterokinase
or TEV protease, the threshold of detection is approximately in
the mid-picomolar to low-nanomolar range, respectively.
6. Initiate the reaction by adding 5–7 μL 1 mM nitrocefin
substrate stock solution to a final concentration of 250 μM
(see Notes 6 and 7).
7. After the addition of nitrocefin, immediately insert the plate
into a plate reader and measure the OD at 492 nm every 2 min.
3.3 Biosensor
Assays
3.3.1 Protease
and MacromoleculeBinding Assay
Sensing by Allosteric Derepression
supernatant into a syringe and attach a 0.45 μm filter to the
end. Filter the supernatant to remove large particles. Replace
the filter if it becomes clogged.
3. Equilibrate a 1 mL Nickel Sepharose His Trap column with
Lysis and Binding buffer by washing the column with 10 column volumes. Load the clarified protein lysate through the
column. Keep an aliquot of the lysate and flow-through for
analysis. Wash the column with 10 column volumes of Lysis
and Binding buffer and collect an aliquot for analysis. Elute
with 10 mL of Elution Buffer. Load equal volume fractions of
all collected aliquots on a SDS-PAGE gel to estimate the efficiency of purification. Add glycerol to a final concentration of
50% and store at −20 °C.
4. Alternatively, if using an FPLC instrument, equilibrate column
with 10 column volumes of Lysis and Binding buffer. Clarified
lysate is loaded into the system at a flow rate of 1 mL/min.
Wash column with 10 column volumes of Lysis and Binding
buffer. Samples are eluted using gradient length of 25. The
entire run can be done at a flow rate of 1 mL/min with the
pressure kept below 0.30 MPa.
1. The assays are generally carried out at room temperature in a
384-well transparent bottom plate.
2. The exact concentration of the sensor and protease or macromolecular binder must be determined empirically. Before starting the assay, test a range of sensor concentrations with varying
concentrations of protease or macromolecular binder to determine the point of optimal sensitivity.
3. When setting up multiple reactions, it is desirable to make a
master mix containing the common components. The typical
reaction in a total volume of 25 μL is set up as follows.
4. Add a suitable volume of PBS that will eventually make up a
total reaction volume of 25 μL (i.e., we add PBS first to prevent small volumes of other reagents from sticking to the side
of the well or remaining stuck to the pipette tip).
5. Add 2 μL of diluted sensor to a final concentration of approximately 5 nM and mix with 2–5 μL of various dilutions of protease
or macromolecular binder. The final optimal concentration of
components needs to be determined empirically. For enterokinase
or TEV protease, the threshold of detection is approximately in
the mid-picomolar to low-nanomolar range, respectively.
6. Initiate the reaction by adding 5–7 μL 1 mM nitrocefin
substrate stock solution to a final concentration of 250 μM
(see Notes 6 and 7).
7. After the addition of nitrocefin, immediately insert the plate
into a plate reader and measure the OD at 492 nm every 2 min.
3.3 Biosensor
Assays
3.3.1 Protease
and MacromoleculeBinding Assay
Sensing by Allosteric Derepression