The TrueBlot® Immunoprecipitation and Western Blot Kit for DYKDDDDK (FLAG®) Epitope Tag contains the critical supporting reagents, buffers, and substrates for immunoprecipitation and Western blotting of samples containing the DYKDDDDK (FLAG®) Epitope Tag using TrueBlot® secondary antibody in conjunction with Rockland's Antibody for the detection of FLAG® conjugated proteins (MOUSE) Monoclonal Antibody.
- The anti-DYKDDDDK (FLAG®) antibody included in this Western blot kit detects FLAG® -tagged recombinant proteins fused with FLAG® tag either at the amino-terminus or carboxy-terminus of the protein.
The FLAG® epitope is a small but highly immunogenic peptide DYKDDDDK (N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C) which allows fusion proteins to retain their original conformation and function. The hydrophilic character of this sequence increases the likelihood that it will be located on the surface of FLAG® -tagged recombinant proteins for reaction with anti-DYKDDDDK (FLAG®) antibodies.
TrueBlot technology enables unhindered detection of protein bands of interest which would otherwise be obscured by the presence of reduced and denatured heavy and light chain immunoglobulin in the blot (as detected by the conventional immunoblotting HRP anti-mouse IgG reagent). Mouse IgG TrueBlot® ULTRA is the unique horseradish peroxidase conjugated anti-mouse IgG immunoblotting secondary antibody which enables detection of immunoblotted target protein bands, without hindrance by interfering immunoglobulin heavy and light chains from your IP antibody. Use it in place of your usual HRP anti-mouse IgG immunoblotting secondary antibody. It is easy to generate publication-quality IP/WB data with Mouse IgG TrueBlot® ULTRA.
Mouse IgG TrueBlot® ULTRA is provided as 1000X solution. In order to conserve reagent, we recommend incubating the blots from minigels in sealed bags (removing as much air as possible) with minimal volume (2-3 mls).
Mouse IgG TrueBlot® ULTRA. An HRP-conjugated second step reagent reacting with mouse IgGs for optimal signal detection in immunoprecipitation/immunoblotting experiments.
Note that there are three key procedural considerations:
1. Protein A or G beads may be used with the mouse, goat and sheep TrueBlot secondaries but not with the rabbit TrueBlot secondary. Use of protein A or G beads with the rabbit TrueBlot will result in contaminating bands.
2. Immunoprecipitate should be completely reduced.
3. Milk should be used as the blocking protein for the immunoblot.
The TrueBlot® Immunoprecipitation and Western Blot Kit for DYKDDDDK (FLAG®) Epitope Tag components are sufficient for 20-25 miniblots.
Components:
1. Mouse IgG TrueBlot ULTRA: 50 μL 18-8817-31
2. TrueBlot Enhancer Solution: 25 mL
3. TrueBlot Blocker: 10 g
4. TrueBlot Assay Buffer: 30 mL 20X
5. TrueBlot Substrate A: 12.5 mL
6. TrueBlot Substrate B: 12.5 mL
7. Anti-Mouse Ig IP Beads: 2.5 mL Binds 0.4 mg Ig/mL beads
8. Antibody for the detection of FLAG™ conjugated proteins (MOUSE) Monoclonal Antibody: 100 µg
9. Western Blot Incubation Tray
Special Notes:
Upon initial use of the IP beads, we recommend that the vial be inverted several times to get the beads into suspension. We recommend to use a large bore pipet to pipet up the liquid for use.
For storage of the opened vial of beads, we recommend that the vial cap be sealed with parafilm to help prevent evaporation of the buffer.
