4 Ligand Screening Studies
Perform a ligand screening study to identify ligand candidates that stabilize a protein
Example experiment files and plate template files are located in the examples folder:
<drive>:\Program Files\Applied Biosystems\Protein Thermal Shift
Software\examples, where <drive> is where you installed the software.
To view the data for the ligand screening example ud in this chapter, u the
Ligand_Screening_Example_ViiA7.eds and
Ligand_Screening_Example_Setup_ViiA7.csv files in the ViiA7 Example Files folder.
page 68)
)
Run the protein melt reactions ()
Set up the analysis (page 72)
Review the well results (Review the replicate results (page 78)
Ligand Screening Studies
General guidelines
4
General guidelines
For general guidelines that apply to all Protein Thermal Shift™ studies, e page9.
Experimental design Before you perform a ligand screening study, we recommend that you first perform a buffer screening study to identify a buffer in which the protein is thermally stable. After you identify ligand candidates, you can perform a ligand titration study to determine the optimal ligand:protein ratio.
Replicates and controls For ligand screening studies, we recommend that you prepare:• At least 4 replicates of each reaction
•At least 4 replicates of no protein controls (NPCs)
•At least 4 replicates of ligand only controls (LOCs)
Create and t up an experiment file for the instrument run
This ction contains general ttings for creating and tting up an experiment file.
For detailed instructions, e:
Experiment file ttings
Instrument Page ViiA™ 7 Real-Time PCR System page12 StepOne™ and StepOnePlus™ Real-Time PCR Systems page14 7500 and 7500 Fast Real-Time PCR Systems page16
Setup Setting Experiment
properties
•Experiment type: Melt Curve
•Reagents: Other
•Ramp speed: Fast or Standard
Target
properties
•Reporter: ROX
•Quencher: None
Plate layout•Assign targets to all wells in u
•Passive reference: None
68Protein Thermal Shift™ Studies Ur Guide
Chapter 4 Ligand Screening Studies
Create and t up an experiment file for the instrument run 4
Defining and
assigning targets We recommend that you define and assign targets so you can review the melt curves for the replicate groups in the Real-Time PCR System Software before importing the
experiment files into the Protein Thermal Shift ™ study. Define the following targets: •One target for each ligand or for each ligand titration
•Define a target for the no protein control (NPC)
•Define a target for the ligand only control (LOC)
IMPORTANT!Make sure that the well assignments correspond exactly with the arrangement of reactions in the reaction plate. Setup errors may result in an incorrect grouping of replicates.
Example targets In the ViiA ™ 7 System ligand screening example file, targets were defined for each
concentration of ligand, the ligand only control (LOC), and no protein control (NPC).
Example plate
谎言歌词谐音layout In this example, rows F–P are not in u. Run method •Reaction Volume Per Well:
20µL复旦大学附属中学
•Thermal profile:
Step 1, Temp: 25°C, Time: 2 minutes
Step 2, Temp: 99°C, Time: 2 minutes
•Ramp mode: Continuous
•Ramp rate:
–ViiA ™ 7 System: Step 1: 1.6°C /s, Step 2: 0.05°C /s
–StepOne ™ and StepOnePlus ™ Systems and 7500 and 7500 Fast
Systems: 1%
•Optical Filters (ViiA ™ 7 System only):
–Excitation Filter: x4(580±10)
–Emission Filter: m4(623±14)
Setup
Setting
Ligand Screening Studies
Prepare the protein melt reactions
4
Prepare the protein melt reactions
Protein melt reaction stability For consistency in Tm values, we recommend that you keep the protein melt reactions on ice until you are ready to load the instrument and start the run.
If the protein is thermally stable at ambient temperatures, you may consider preparing the reactions i
n advance and leave the reaction plate at ambient temperature, protected from light. However, the fluorescence levels will decrea over time and the Tm values will vary, depending on the protein and its thermal stability. If you want to prepare the reaction plates in advance, we recommend that you first determine the benchtop stability of your protein melt reactions.
Required materials Required materials for protein melt reactions:
•Protein Thermal Shift™ Dye (1000✕)
•Protein Thermal Shift™ Buffer
•Water
•Protein
•Ligands or ligand titrations
•MicroAmp® Optical Reaction Plate appropriate for your real-time PCR
instrument
•MicroAmp® Optical Adhesive Film appropriate for your reaction plate
Prepare the protein melt reactions We recommend that you prepare four replicates of each reaction.
1.Prepare a fresh dilution of Protein Thermal Shift™ Dye (1000✕) to 8✕.
2.Place the appropriate reaction plate or tubes on ice, then prepare the protein melt
reactions:
get fresh•Make sure that the arrangement of reactions in the reaction plate corresponds exactly with the well assignments in the experiment file.
IMPORTANT!Setup errors may result in an incorrect grouping of the data
and incorrect Tm statistics.
•Add reaction components to the plate in the order listed.
3.Pipet each reaction up and down 10 times to mix well.
4.Seal the plate with MicroAmp® Optical Adhesive Film, spin it at 1000rpm for
1minute, then place it on ice.
Component Volume
Protein Thermal Shift™ Buffer 5.0µL
Water+protein +ligand12.5µL
Diluted Protein Thermal Shift™ Dye (8✕) 2.5µL
Total volume for each control reaction20.0 µLdnk
70Protein Thermal Shift™ Studies Ur Guide
Chapter 4 Ligand Screening Studies
Run the protein melt reactions
4 Run the protein melt reactions
Load and run the protein melt reactions on a supported Applied Biosystems
getoffReal-Time PCR System, then analyze and save the experiment file before importing the
experiment file into the Protein Thermal Shift™ Software.
六级真题下载IMPORTANT!Keep the protein melt reactions on ice until you load the instrument.
Load and run the reactions 1.If necessary, transfer the experiment file that you created for the run to the
computer that is connected to the instrument.
2.In the Home screen of the Real-Time PCR System Software, click Open, then
lect the experiment file you created for the run.temporary是什么意思
3.Load the reaction plate into the instrument.
4.In the Real-Time PCR System Software, click Run in the navigation pane, then
click START RUN.
Review the melt curves Using the Real-Time PCR System Software, open the experiment file from the completed instrument run, analyze and save the experiment file, then review the melt curves.
Note:You must analyze and save the experiment file in the Real-Time PCR System Software before you can import it into the Protein Thermal Shift™ Software.
Some common troubleshooting caus are provided here. For more detailed troubleshooting information, e page83.
1.In the Home screen of the Real-Time PCR System Software, click Open, then
lect the experiment file from the instrument run.
2.View the melt curves:
3.Review the melt curves:
•Do you e fluorescence signals in all of the sample wells?
No fluorescence signals in the sample wells may indicate missing dye or
protein or an instrument problem.
•Do you e flat fluorescence levels in the NPC wells?
High fluorescence levels in the NPC wells may indicate protein
contamination in the wells or protein melt reactions; or it may indicate that
the dye interacts with a component in the buffer.
Real-time PCR
System Software
View the melt curve
ViiA™ 7 Software Click Analysis > Melt Curve Plot in the navigation pane,
lect Normalized Reporter from the Plot dropdown list,
then lect Target from the Color dropdown list.
StepOne™ Software or
7500 Software
Click Analysis > Melt Curve in the navigation pane, lect
Normalized Reporter from the Plot dropdown list, then
lect Target from the Color dropdown list.
Ligand Screening Studies
Set up the analysis
4
•Do you e flat fluorescence levels in the LOC wells?
High fluorescence levels in the LOC wells but not in the NPC wells may
leviindicate protein contamination in the ligand or ligand-dye interactions.
Fluorescence from ligand-dye interactions may mask the protein-dye
interactions.
•Do the replicates have similar melt curves?
4.Save, then clo the experiment file.
Note:The melt curves in the real-time PCR software may not exactly match the melt
curves in the Protein Thermal Shift™ Software. When the experiment files areiperf
imported into the Protein Thermal Shift™ Software, the Protein Thermal Shift™
Software reduces the noi in the fluorescence data.
Example melt
curves
Melt curves for the ligand screening example file from the ViiA™ 7 System:
Set up the analysis
This ction provides instructions for tting up the protein thermal shift study using
Protein Thermal Shift™ Software v1.0. For more information about how to u the
software, refer to the Protein Thermal Shift™ Software Help.
Setup guidelines•The experiment files that you import into the study must contain analyzed melt
curve data from a complete melt curve run.
•Set up the analysis group so that it contains experiment files from only one
instrument.
Create and t up the study 1.In the Home screen of the Protein Thermal Shift™
Software, click Create Study.
2.Complete the Setup > Properties screen:
•The Study Name cannot be more than 100 characters and cannot contain the characters: / \ * “ ? < > | . ,
•The instrument lection must match the instrument type that you ud to run the protein melt reactions and generate the experiment files.
新动力英语72Protein Thermal Shift™ Studies Ur Guide
