AntiGFP,Monoclonal Antibodies
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Green fluorescent protein (GFP) from jellyfish Aequorea victria has been used with increasing success as a marker
protein for gene expression, intracellular processes and localization ranging from bacteria to transgenic mice.
Wako offers mouse monoclonal antibodies applicable for various purposes.
Clone No. Application Wako Cat. No. (Pkg. Size)
Western blotting Immunoprecipitation Immunostaining
mFX75 unconfirmed unconfirmed            012-22541    (100 μL)
mFX73            012-20461    (100 μL)
           018-20463    (100 μL 5)
for Western blotting
Anti Green Fluorescent Protein, Monoclonal Antibody (mFX75)
Clone No. mFX75
Preparation Prepared by a hybridoma, which is established by fusing mouse myeloma cells, NSO and spleen cells of BALB/c
mice immunized with the the GFPs partial peptide coupled with BSA after being immunized with BSA after being
immunized with GFP. The hybridoma supernatant cultured by Hollow fiber instrument was purified by Protein A
affinity chromatography.
Subclass mouse IgG2a·κ
Concentration shown on each label (approximately 1 mg/mL)
Appearance D-PBS(-) containing 10 mmol/L Hepes (pH 7.4) and 10 mmol/L Sodium Azide
Working Dilution 1: 500 ~ 1: 1,000
C           : HepG2 cell Lysate (5104 cells)
Lane 1 : HepG2 cell Lysate (5104 cells) + rGFP 1 ng
Lane 2 : HepG2 cell Lysate (5104 cells) + rGFP 10 ng
HepG2 cell Lysate with 1 ng or 10 ng of recombinant GFP
protein was subjected to SDS-PAGE, transferred to PVDF
membrane, and exposed to X-ray film. Exposure time: 3 min.
Monoclonal antibodies supplied from Wako and manufacturer
S and N were used as primary antibodies at final
concentration of 1 μg/mL.
for Immunoprecipitation & Immunostaining
Anti Green Fluorescent Protein, Monoclonal Antibody (mFX73)
Clone No. mFX73
Preparation Prepared by a hybridoma, which is established by fusing mouse myeloma cells, NSO and spleen cells of
BALB/c mice immunized with the GFPs partial peptide coupled with BSA after being immunized with GFP.
The hybridoma supernatant cultured by protein-free culture was subjected to ultrafiltration with an ultrafilter
30,000-molecular-weight cutoff.
Subclass mouse IgG2a·κ
Concentration shown on each label (approximately 1 mg/mL)
Appearance PBS containing 10 mmol/L Hepes (pH 7.4) and 10 mmol/L Sodium Azide
Working
Concentration
Immunoprecipitation      1 ~ 5 μg/5 μL Sepharose
Immunostaining              0.5 ~ 1 μg/mL
Binding with GFP in immunoprecipitation assay
SDS-PAGE Silver Staining
Sample: applied GFP (0.5 μg/10 μL) and each eluent (10 μL)
Gel: SuperSep 10-20%, 17 well (Wako Cat. #191-13071)
Detection Reagent: Silver Stain II Kit Wako (Wako Cat. #291-50301)
Lane 1: applied GFP
Lane W,M,B: eluent from each anti-GFP antibody solid-phased support.
                       (Wako's anti-GFP antibody (mFX73),
                       manufacturer M's and B's antibodies are used,respectively.)
Anti-GFP antibodies supplied from each manufacturer were attached to a solid-
phase of protein G Sepharose and binding with GFP were compared using an
immunoprecipitation assay.
Wakos antibody (mFX73) bound to GFP with higher affinity than antibodies
supplied from manufacturers M and B.
Double staining with anti-serotonin antibody in
transgenic C. elegans
C. elegans strain: unc-18(e81) ; unc-18::EGFP, L4-stage larvae
Fixation: o/n fixation in 4% paraformaldehyde / 1% glutaraldehyde at 4C
Primary antibody: Anti-GFP antibody (mFX73) 1 μg/mL + Anti-Serotonin serum (1:500)
Secondary antibody: Cy3 conjugated Anti-mouse antibody (1:200) + Biotin-conjugated
                                  Anti-rabbit IgG (1:200) / ABC complex (1:100)
Detection: DAB / Fluorescence
A few serotonin-positive neurons and many cell bodies and dendrites of neurons are stained.
(Data provided by: Dr. Shohei Mitani, associate professor at Tokyo women's medical university)
Product List
Description Wako Cat. No. Package Size
Anti Green Fluorescent Protein, Monoclonal Antibody (mFX75) 012-22541      100 μL
Anti Green Fluorescent Protein, Monoclonal Antibody (mFX73) 012-20461
018-20463
     100 μL
     100 μL 5

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