- FLISA, Flow Cytometry (FACS), Fluorescence Microscopy (FM), Immunohistochemistry (IHC), Western Blotting (WB)
Synonyms: FITC, TrueBlot, FITC TrueBlot ULTRA, Fluorescein TrueBlot, TrueBlot for IP/WB, TrueBlot for immunoprecipitation, TrueBlot for western blotting, Fluorescent TrueBlot, Rb TrueBlot
Background: Rabbit IgG TrueBlot® is a unique fluorescein conjugated Anti-rabbit IgG immunoblotting (second step) reagent. Rabbit IgG TrueBlot® enables detection of immunoblotted target protein bands, without hindrance by interfering immunoprecipitating immunoglobulin heavy and light chains. It is easy to generate publication-quality IP/Fluorescent Western Blot data with Rabbit IgG TrueBlot®, simply substitute the conventional FITC Anti-rabbit IgG blotting reagent with Fluorescent Rabbit TrueBlot® Antibody Fluorescein and follow the prescribed protocol for sample preparation and immunoblotting. Rabbit IgG TrueBlot® is ideal for use in protocols involving immunoblotting of immunoprecipitated proteins. TrueBlot preferentially detects the non-reduced form of rabbit IgG over the reduced, SDS-denatured form of IgG. When the immunoprecipitate is fully reduced immediately prior to SDS-gel electrophoresis, reactivity of Rabbit IgG TrueBlot® with the 55 kDa heavy chains and the 23 kDa light chains of the immunoprecipitating antibody is minimized thereby eliminating interference by the heavy and light chains of the immunoprecipitating antibody in IP/Western blot applications. Applications include studies examining post-translational modification (e.g., phosphorylation or acetylation) or protein-protein interactions.
- Fluorescent Rabbit TrueBlot® Antibody Fluorescein Conjugate was prepared from tissue culture supernatant by Protein G affinity chromatography. Assay by Immunoelectrophoresis resulted in a single precipitin arc against anti-fluorescein and Anti-Rabbit Serum. Reactivity is observed against native Rabbit IgG by both Western blot and ELISA.
- Labeling Ratio
- Application Notes
Immunohistochemistry Dilution: User Optimized
Application Note: Fluorescein Conjugated Antibodies are designed for immunofluorescence microscopy, fluorescence based plate assays (FLISA) and fluorescent western blotting. This product is also suitable for multiplex analysis, including multicolor imaging, utilizing various commercial platforms. Fluorescent Rabbit TrueBlot® Antibody Fluorescein may also be used for detection in immunoassays that do not employ immunoprecipitation. Fluorescent Rabbit TrueBlot® Antibody Fluorescein is provided as a lyophilized powder. 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). If used conservatively at 2.5 mLs/blot will yield enough reagent for 40 blots. Note that there are three key procedural considerations: 1. Protein A or G should not be used for the immunoprecipitation. Use of protein A or G beads with the rabbit TrueBlot will result in contaminating bands. For immunoprecipitation, Anti-rat IgG beads or Anti-rabbit IgG beads should be used for rat or rabbit immunoprecipitating antibodies, respectively. 2. Immunoprecipitate should be completely reduced. 3. MB-070 Blocking Buffer for Fluorescent Western Blotting should be used as the blocking protein for the immunoblot. All recommended dilutions for listed applications are intended as an initial recommendation, specific conditions for each protein and antibody combination should be specifically optimized by the end user.
FLISA Dilution: User Optimized
Flow Cytometry Dilution: 1:2,000 - 1:10,000
Western Blot Dilution: 1:1000
IF Microscopy Dilution: 1:500 - 1:2,500
- For Research Use only
Reconstitution Volume: 100 μL
Reconstitution Buffer: Restore with deionized water (or equivalent)
Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Stabilizer: 10 mg/mL Polyethylene Glycol (PEG-8000)
- Sodium azide
- Precaution of Use
- This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
- RT,4 °C,-20 °C
- Storage Comment
- Store vial at 4 °C prior to restoration. For extended storage aliquot contents and freeze at -20 °C or below. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4 °C as an undiluted liquid. Dilute only prior to immediate use.
- Expiry Date
- 12 months