rev: March 26, 2004
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(anti-Human and others as indicated)
RDI Division of Fitzgerald Industries Intl offers a wide line of antibodies. Since no one antibody works best for all applications (neutralization, blotting, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.
cat#RDI-GRNFP3abG $500.00/1mg (unconjugated)
cat#RDI-GRNFP3abg-HRP $562.00/1mg (HRP conjugated)
cat#RDI-GRNFP3abG-FT $500.00/1mg FITC conjugated
cat#RDI-GRNFP3abG-BT $500.00/1mg Biotin conjugated
cat#RDI-GRNFP3abG-ALK $625.00/1mg Alk Phos conjugated
cat#RDI-GRNFP3abG-TX $562.00/1mg Texas Red conjugated
-affinity purified anti-GRNFP can be used to detect GFP an its variants in ELISA, immunoblotting and immunoprecipitation.
cat#RDI-GRNFP3abm $625.00/1mg (unconjugated)
cat#RDI-GRNFP3abm-HRP $688.00/1mg (HRP conjugated)
cat#RDI-GRNFP3abm-FT $688.00/1mg FITC conjugated
cat#RDI-GRNFP3abm-BT $688.00/1mg Biotin conjugated
cat#RDI-GRNFP3abm-ALK $688.00/1mg Alk Phos conjugated
cat#RDI-GRNFP3abm-TX $6881.00/1mg Texas Red conjugated
- purified moncolonal anti-GRNFP can be used to detect GFP an its variants in ELISA, immunoblotting and immunoprecipitation.
This polyclonal antibody was generated using E. coli-expressed full-length GFP (green fluorescent protein) as an immunogen. This antibody reacts with wild-type GFP,and its variants EGFP and EBFP. The tested titer for Western blot is 1:5,000 in wtesern blot
ackground: Since the detection of intracellular Aequorea Victria Green
Fluorescent Protein (GFP) requires only irradiation by UV or blue light,
it provides an excellent means for monitoring gene expression and protein
localization in living cells. This monoclonal can be used to detect GFP and
its variants in immunoblotting and immunoprecipitation.
Packaging: lyophilized antibody. Reconstitute with 0.1ml distilled water to yield 1.0mg/ml IgG in PBS pH 7.2 with 1% sucrose and 0.1% sodium azide. Allow to set at leats 30 minutes, mix well.
Immunogen: GST-GFP fusion protein corresponding to full length amino acids (246aa) of GFP
Hybridoma: mouse myeloma cell P3U1 with Balb/c splenocyte
Purification: Protein A sepharose of mouse ascites
Specificity: This antibody detects GFP tagged protein on:
-western blot (approx 1ug/ml )
-also reacts with EGFP, pS65T-c1 and pRS-GFP.
ref: Cubitt A.B. et al. Trends Biochem Sci 20, 448 (1995)
Chalfie M. et al. Science 263, 802 (1994)
Storage: Lyophilized antibody is stable for at least 1 year from date of shipment when stored at 4 DEG C. Reconstituted antibody should be stored at 4 DEG C short term or aliquoted and frozen at -20 DEG C. Avoid frequent freeze thaw cycles.
For In Vitro Research Use Only
1. Boil all samples for 3-5 minutes. Load 10 ul of cell lysate or tissue homogenate (5-20 ug total protein) to each well of an SDS- polyacrylamide gel and electrophorese in a 1 mm thick gel.
2. Transfer to a a polyvinylidene difluoride (PVDF) membrane at 10 V for 1 hour with semi-dry transfer system. (Transfer Buffer:23mM Tris, 190 mM glycine, 20% MeOH).
3. The transferred proteins can be visualized by staining the membrane for 1 minute with Ponceau S (SIGMA CAT#: P-7170). Rinse the membrane with PBS.
4. Non-specific binding sites are blocked by immersing the membrane in 5% Skim Milk/PBS/0.05% for 1 hour at 37 DEG C or overnight at 4'C.
5. Incubate in primary antibody for 1 hour at room temperature. (The concentration of the antibody to be used will depend on several variables and the abundance of the antigen).
6. Wash the membrane 3 times with PBS, 0.2% Tween20 for 5-10 minutes per wash.
7. Incubate in Horseradish Peroxidase conjugated anti-rabbit diluted 1:3000 * in PBS, for 45 minutes at room temperature. * or as recommended by the manufacturer.
8. Wash the membrane 3 times with PBS, 0.2% Tween20 for 10 minutes per wash.
9. Incubate in Amersham ECL Reagent for 1 minute. Drain membrane, remove excess ECL Reagent by dabbing with a Kimwipe, and seal in plastic wrap.
10.Expose to ECL hyperfilm in a dark room for 30 seconds.Develop as usual for autoradiogram or X-ray. The condition for develop-nnt and exposure may vary.
1. Rinse the cells twice with PBS, then lyse the cells with the addition of 1.2 ml of cold Lysis buffer (50mM Hepes ph 7.2-7.6, 250mM NaCl, 0.1% NP-40, 5mM EDTA, 10% glycerol), vortex well and put thecells on ice for 30 minutes.
2. Remove the lysate by centrifugation the cells at 12000 x g for 20 min. at 4'C.
3. Add the antibody at the amount of as suggest in APPLICATIONS to the supernatant containing approximately 100-500 ug total protein, vortex and incubate with gently agitation on ice for 30 minutes.
4. Add 40 ul of 50% ProteinA-Sepharose, vortex and incubate with gently agitation on ice for one hour.
5. Wash five times by centrifugation at 2500 x g for 10 seconds with Lysis buffer
6. Dissolve the pellet in 40 ul of Laemli SDS sample buffer, boil for 3-5 minutes, and centrifuge at 3000 x g for 5 minutes. Load 10-20 ul of supernatant on to the gel and electrophorese normally. Transfer to PVDF membrane and probe with appropriate antibodies.
For cultured cell: Immunoperoxidase staining Fixing:
1. Rinse the cells on glass coverslips in phosphate buffered saline (PBS), and immerse for 15 minutes in 4% paraformaldehyde diluted in PBS. Then rinse the coverslips 2 times for 5 minutes each in PBS. Blocking:
1. Cover the cells with 3% H2O2 in PBS for 10 minutes at room temperature. Then rinse the coverslips 2 times for 5 minutes each in PBS.
2. Cover the cells with 10% normal goat serum (NGS) in PBS for 15 minutes to minimize non-specific adsorption of the antibodies to the cover slip (2-50ul is usually sufficient).
1. Remove the blocking buffer.
2. Incubate in primary antibody at the concentration of as suggest in APPLICATIONS diluted in 1% NGS in PBS for 1 hour at room temperature. (The concentration of antibody to be used will depend on several variables and the abundance of the antigen).
3. Wash the cell 3 times in PBS for 5 minutes each.
4. Incubate in Polyvalent Biotinylated Secondary Antibody for 10 minutes at room temperature. (OR CAN USE ANTI-MOUSE:rHODAMINE CONJUGATE FOR FLUORESCENCE DeTECTION)
5. Wash the cell 3 times in PBS for 10 minutes each.
6. Incubate in Streptavidin-Peroxdase Reagent for 10 minutes at room temperature.
7. Wash the cell 3 times in PBS for 5 minutes each.
8. Visualize with DAB for approximately 1-8 minutes.
Wash in distilled water.
9. Counterstain in hematoxylin. Wash 3 times in distilled water.
-See other epitope tagging antibodies including maltose binding protein, HA-Tag, Myc-Tag, VSV-G-Tag, Thyoredoxin, SjGst , Green Fluorescent Protein , GLU-GLU, AU1 , AU5 and more.
RDI Division of Fitzgerald Industries Intl
34 Junction Square Drive
Concord MA 01742-3049
phone (800) 370-2222
or (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
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