anti pp1β  (Santa Cruz Biotechnology)

Journal: STAR Protocols

Article Title: Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates

doi: 10.1016/j.xpro.2024.103328

Figure Lengend Snippet: Testing the performance of a sensor Plate-maps illustrating the workflow for testing sensor performance. The steps of dispensing LgBiT-MYPT1 ( ) and SmBiT-PP1β ( ) lysate or buffer ( ) are indicated, and the luciferase signal of a corresponding time-course experiment, showing the association of the SmBiT-PP1β:LgBiT-MYPT1 sensor. The signal and background levels are indicated.

Article Snippet: Synthetic C terminus of human PP1β (residues 298–327) , GenScript , NA.

Techniques: Luciferase

Journal: STAR Protocols

Article Title: Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates

doi: 10.1016/j.xpro.2024.103328

Figure Lengend Snippet: Optimizing assay sensitivity by 2D matrix titration of lysates Plate-maps illustrating the positions for making the dilution series of SmBiT-PP1β ( ) and LgBiT-MYPT1 ( ) lysates in a 96-well plate (Step 1) and for dispensing the dilution series as a 2D matrix in a 384-well plate (Step 2 + 3). Note that the signal and corresponding background wells are marked for the calculation of signal-to-background (S/B) values. The lower left panel shows an example result with the average S/B for each condition plotted and colored to produce a heat-map that indicates the conditions with highest (dark blue) S/B values. In Step 4 a plate map indicates the positions for spiking with a positive competitor ( ) and negative control ( ) and the lower right panel shows the % modulation by the positive control with respect to the negative control. The yellow and green squares indicate the optimal working dilutions for the corresponding assay (HTS or time course competition assay).

Article Snippet: Synthetic C terminus of human PP1β (residues 298–327) , GenScript , NA.

Techniques: Titration, Negative Control, Positive Control, Competitive Binding Assay

Journal: STAR Protocols

Article Title: Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates

doi: 10.1016/j.xpro.2024.103328

Figure Lengend Snippet: Protocol steps for performing a time-course competition assay Plate-maps illustrating the workflow for performing a time-course competition assay, dispensing LgBiT-MYPT1 ( ) lysate (Step 1) and SmBiT-PP1β ( ) lysate (Step 2), and the subsequent competition (Step 3) with 100 μM C-terminus of PP1β (residues 298–330) ( ) or 100 μM 18R1K7 ( ), alongside the vehicle control ( ). The lower panel shows the corresponding luciferase signal over time.

Article Snippet: Synthetic C terminus of human PP1β (residues 298–327) , GenScript , NA.

Techniques: Competitive Binding Assay, Control, Luciferase

Journal: STAR Protocols

Article Title: Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates

doi: 10.1016/j.xpro.2024.103328

Figure Lengend Snippet: Modulators of interactions between PP1/PP2A subunits that can be used for time-course competition assays

Article Snippet: Synthetic C terminus of human PP1β (residues 298–327) , GenScript , NA.

Techniques: Control, Purification, Recombinant, Binding Assay, Bioprocessing, Activity Assay

Journal: STAR Protocols

Article Title: Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates

doi: 10.1016/j.xpro.2024.103328

Figure Lengend Snippet: Characterization of the binding behavior of the PP1:PPP1R2 complex Time-course competition assay with a LgBit-PP1:R2-SmBiT sensor. All competitors were applied at 10 μM. CD: NIPP1 central domain (residues 143–224); MC: microcystin.

Article Snippet: Synthetic C terminus of human PP1β (residues 298–327) , GenScript , NA.

Techniques: Binding Assay, Competitive Binding Assay

Journal: STAR Protocols

Article Title: Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates

doi: 10.1016/j.xpro.2024.103328

Figure Lengend Snippet: Characterization of the binding behavior of the PP1:SDS22 complex Time-course competition assay with a LgBit-PP1:SDS22-SmBiT sensor. All competitors were applied at 10 μM. CD: NIPP1 central domain (residues 143–224); MC: microcystin.

Article Snippet: Synthetic C terminus of human PP1β (residues 298–327) , GenScript , NA.

Techniques: Binding Assay, Competitive Binding Assay

Journal: STAR Protocols

Article Title: Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates

doi: 10.1016/j.xpro.2024.103328

Figure Lengend Snippet: Measurement of the stimulation of PP1:SHOC2 complex formation by different concentrations of the modulator GTP-MRAS

Article Snippet: Synthetic C terminus of human PP1β (residues 298–327) , GenScript , NA.

Techniques:

Journal: STAR Protocols

Article Title: Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates

doi: 10.1016/j.xpro.2024.103328

Figure Lengend Snippet: Examples of troubleshooting data (A) Example of decreasing luciferase activity over time, for an assay where the luciferase activity is too high (blue) and causes substrate depletion and for an assay with acceptable luciferase activity showing normal signal decay (purple, t1/2 = 45–60 min) (See ). Both traces are standardized to 100% for comparison. (B) Example illustrating how different purification preps of the same competitor can exhibit varying degrees of modulation of a sensor, the arrow indicates when the competitor was added (See ). (C) Example demonstrating the validation of competitor protein by testing the same protein preparation on a different sensor, the arrow indicates when the competitor was added. (See ). (D) Example illustrating that for some competitors it is beneficial to use full-length protein to effectively modulate a sensor. Here, His-R2 variants were used to dissociate a PP1:R2 sensor, only full length (FL) R2 could effectively dissociate the sensor, not the truncation mutants ΔN (R2-116-205) and ΔC (R2-1-123). The arrow indicates when the competitor was added. (See ). (E) Example showing that high concentrations (>100 μM) of competitor protein or peptide might be necessary for complete dissociation of a sensor (See ). (F) Example illustrating that preincubation with competitor protein before sensor formation can more effectively modulate a sensor compared to competition when the sensor is formed, the arrow indicates when the competitor was added (See ). (G) Example of assay interference, marked by sudden drops in luciferase signal following addition (arrows) of various components to the plate (See ). (H) Example demonstrating how temperature changes of the plate impact luciferase signal readout (See ).

Article Snippet: Synthetic C terminus of human PP1β (residues 298–327) , GenScript , NA.

Techniques: Luciferase, Activity Assay, Comparison, Purification, Biomarker Discovery

Journal: STAR Protocols

Article Title: Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates

doi: 10.1016/j.xpro.2024.103328

Figure Lengend Snippet:

Article Snippet: Synthetic C terminus of human PP1β (residues 298–327) , GenScript , NA.

Techniques: Virus, Recombinant, Software