BromoCatch™ Ligands Usage Protocols

Tips:

• Spin down contents before opening
• Store reconstituted stock at –20 °C protected from light
• Avoid repeated freeze–thaw cycles
• BromoCatch™ Control Ligand can be used at X concentration to compete with other BromoCatch™ probes in control experiments

Cell lysate labeling Protocol

Example data generated using H2B, generalizable protocol for your protein of interest (POI).

Materials & Reagents

• HEK293-FT cells
• pCMV POI-BromoCatch vector
• DMEM, Optimem, FBS (10%)
• TAMRA, BromoCatch™ Ligand (Cat. No. 8938)
• DMSO (control)
• RIPA buffer
• Primary anti-H2B antibody (Rabbit)
• Secondary antibody (IR800)
• 6 or 12 well plates
• Humidified incubator (5% CO₂, 37°C)
• Trypsin
• Western blot reagents

Protocol

1. Transfection: Transfect HEK293-FT cells with the pCMV POI-BromoCatch vector using standard transfection reagents and incubate in a humidified incubator (5% CO₂, 37°C) for at least 16 hours.
2. Cell Plating: Trypsinize transfected cells and plate them onto 6 or 12 well plates and allow cells to adhere for 16 hours.
3. Treatment with Probe: Replace DMEM with Optimem + 10% FBS, containing either DMSO (control) or increasing concentrations of TAMRA, BromoCatch™ Ligand.
4. Incubation: Incubate treated cells for 2 hours in a humidified incubator (5% CO₂, 37°C).
5. Cell Washing: Gently wash cells with warm Optimem + 10% FBS media to remove excess probe.
6. Cell Lysis: Lyse cells using RIPA buffer to extract proteins.
7. Western Blotting & Membrane Transfer: Perform SDS-PAGE and transfer proteins onto a membrane.
8. Fluorescence Imaging: Directly image the membrane using the TMR fluorescence channel.

Control: POI Antibody Staining (if required)

      9. Primary Antibody Staining: Incubate membrane with anti-H2B antibody.

    10. Secondary Antibody Staining: Apply IR800-conjugated secondary antibody for detection.

Figure 1. TAMRA probes for cell lysate experiments.

TAMRA, BromoCatch™ Ligand (Cat. No. 8938) specifically detects H2B-BromoCatch at 0.25 μM concentration of probe. The probe showed no unspecific binding in HEK293FT WT cells when incubated at up to 2.5 μM.

Live-Cell Labeling Protocol (Fluorescent Probes)

Materials & Reagents

• U2-OS cells
• pCMV POI-BromoCatch vector
• DMEM medium
• Optimem + 10% FBS
• Humidified incubator (5% CO₂, 37°C)
• DMSO
• Janelia Fluor® 635, BromoCatch™ Ligand (Cat. No. 8937)
• Hoechst 33342
• Trypsin
• Confocal microscope

Protocol

1. Transfection: Transfect U2-OS cells with pCMV POI-BromoCatch vector using a suitable transfection reagent and incubate the cells in a humidified incubator (5% CO₂, 37°C) for at least 16 hours.
2. Cell Plating: Trypsinise the transfected cells and plate them onto microscopy slides and allow cells to adhere for 16 hours under standard incubation conditions.
3. Treatment with Fluorogenic Probe: Replace DMEM medium with Optimem + 10% FBS, supplemented with DMSO (control) or 200 nM Janelia Fluor^® 635, BromoCatch™ Ligand.
4. Probe Incubation: Incubate cells with the probe for up to 8 hours in a humidified incubator (5% CO₂, 37°C).
5. Cell Washing & Staining: Gently wash cells 3 times with warm Optimem + 10% FBS and add Hoechst 33342 to each well and incubate for 30 minutes.
6. Imaging: Perform confocal microscopy imaging to analyze probe fluorescence and nuclear staining.

Figure 2: Cellular validation of Janelia Fluor^® 635, BromoCatch™ Ligand using live-cell confocal microscopy.

Pulldown Protocol (Biotin BromoCatch™ Ligand)

Materials & Reagents

• HEK293-FT cells
• pCMV H2B-BromoCatch vector
• DMEM, Optimem, FBS (10%)
• Biotin BromoCatch™ Ligand (Cat. No. 8939)
• DMSO (control)
• RIPA buffer
• Primary anti-POI antibody
• Secondary antibody (IR800)
• 6 or 12 well plates
• Humidified incubator (5% CO₂, 37°C)
• Trypsin
• Western blot reagents

Protocol

1. Transfection: Transfect HEK293-FT cells with the pCMV H2B-BromoCatch vector using standard transfection reagents and incubate in a humidified incubator (5% CO₂, 37°C) for at least 16 hours.
2. Cell Plating: Trypsinize transfected cells and plate them onto 6 or 12 well plates and allow cells to adhere for 16 hours.
3. Treatment with probe: Replace DMEM with Optimem + 10% FBS, containing either DMSO (control) or increasing concentrations of Biotin BromoCatch™ Ligand.
4. Incubation: Incubate treated cells for 2 hours in a humidified incubator (5% CO₂, 37°C).
5. Cell Washing: Gently wash cells with warm Optimem + 10% FBS media to remove excess probe.
6. Cell Lysis: Lyse cells using RIPA buffer to extract proteins.
7. Western Blotting & Membrane Transfer: Perform SDS-PAGE and transfer proteins onto a membrane.
8. Streptavidin TMR antibody incubation: Image membrane using the TMR fluorescence channel.​

Control: POI Antibody Staining (if required)

      9.  Primary Antibody Staining: Incubate membrane with Rabbit anti-H2B antibody.

     10. Secondary Staining: Apply IR800-conjugated secondary antibody for detection.

Figure 3: Biotin probes for cell lysate experiments.

Biotin BromoCatch™ Ligand (Cat. No. 8939) specifically detects H2B-BromoCatch at 0.25 μM concentration of probe. The probe showed no unspecific binding in HEK293FT WT cells when incubated at up to 2.5 μM.

Click Chemistry Protocol (Alkyne BromoCatch™ Ligand)

1. Label cells or protein with 250 nM–1 µM ligand.
2. Perform CuAAC with azide-fluorophore, CuSO4, ligand, and sodium ascorbate.
3. Incubate 30 min at RT, then wash thoroughly.
4. Proceed to analysis depending on azide used (e.g., fluorescence etc_)

Plasmids – coming soon

To streamline adoption of the BromoCatch™ platform, we have a comprehensive suite of ready-to-use plasmids for mammalian expression of BromoCatch tagged proteins. These constructs are optimized for flexible cloning and high-level expression in a range of cell types, supporting both N- and C-terminal fusions to your protein of interest.

Each vector includes a BromoCatch domain flanked by flexible glycine-serine linkers (GSL) and multiple cloning sites, enabling modular insertion of target sequences. Options are available with or without N- or C-terminal His-tags for affinity purification, and with your choice of CMV or TK promoters for high or moderate expression, respectively. Vectors are available with puromycin or hygromycin B selection markers to suit diverse experimental workflows.

Whether you're performing live-cell imaging, pull-down assays, or proximity labeling, these plasmids provide a reliable and efficient starting point for generating BromoCatch tagged fusion proteins.