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GFP | Green Fluorescence Protein

AS20 4443 | Clonality: Polyclonal |  Host: Rabbit | Reactivity: Green Fluorescence Protein (GFP)

GFP | Green Fluorescence Protein in the group Tag Antibodies / GFP/YFP/RFP/CFP at Agrisera AB (Antibodies for research) (AS20 4443)
GFP | Green Fluorescence Protein



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Product Information

Immunogen Recombinant GFP protein derived from Aequorea victoria, UniProt: P42212
Host

Rabbit

Clonality Polyclonal
Purity Immunogen affinity purified serum in PBS pH 7.4.
Format Lyophilized
Quantity 50 ĩg
Reconstitution For reconstitution add 50 µl of sterile water
Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
Tested applications Chromatin Immunoprecipitation (ChIP), Immunoprecipitation (IP), Western blot (WB)
Recommended dilution 1: 5000 - 1 : 10 000 (WB)

Reactivity

Confirmed reactivity Recombinant GFP overexpressed in E.coli, from: Arabidopsis thaliana, Chlamydomonas reinhardii, Nicotiana tabacum

Application examples

Application examples
Western blot using rabbit anti-GFP antibodies

194 µg/well of total protein extracted freshly from tobacco with write exact buffer components (50mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% Glicerol, 1 mM DTT, 1X Pefablock) and denatured with SDS 2% and 0.1% Bromophenol blue (Laemmli buffer) at 95°C for 5 min.... were separated on 12 % SDS-PAGE and blotted 2hs to PVDF/nitrocellulose (pore size of um), using wet transfer. Blot was blocked with 5% milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 in TBS-T ON/4°C (with or without milk) with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:20 000 in for 1h/RT with agitation. The blot was washed as above and developed for 2 min with Agrisera ECLSuperBright with name and supplier. Exposure time was 60 seconds.

Courtesy of Delfina Gagliardi Instituto de Agrobiotecnología del Litoral (IAL), Argentina


Immunofluorescence on plant tissue using anti-GFP polyclonal antibodies

Super-resolution image of immunofluorescent localization of GFP-fused protein in Nicotiana tabacum (tobacco) leaf tissue using Agrisera anti-GFP antibodies (AS20 4443) and donkey anti-rabbit IgG DyLight 650 conjugated secondary antibodies (Green). Calcofluor White (Blue) and Tissue Autofluorescence (Yellow),

Method:
Fixation: 2% paraformaldehyde + 0.5% glutaraldehyde, dehydration in ethanol, embedment and UV polymerization in HM20 resin
Cell wall digestion: no
Membrane permeabilization: DMSO-IGEPA
Air drying; No
Antigen retrieval: No
Blocking buffer: 4% w/v non-fat milk powder in 1X Phosphate Buffered Saline buffer, prepared freshly, filtered with 0.2 µm filer
Washing buffer: 1X Phosphate Buffered Saline buffer Primary antibody dilution and incubation time: 1:100; 1 hour at room temperature
Secondary antibody dilution and incubation time and supplier: 1:200 Donkey anti-rabbit IgG (H&L) DyLight®650 (Agrisera, AS12 2254); 1 hour at room temperature
Co-staining of the nucleus (DAPI):  No
Cell wall and nucleus staining: Calcofluor White
All material was freshly prepared. All incubation volumes were kept the same: 200 μL per 22x22 mm cover glass/sample

Courtesy Dr. Kirk Czymmek, Donald Danforth Plant Science Centre, USA

Additional information

Related products

Background

Background GFP (Green fluorescent protein) was originally identified in photo organs on jellyfish Aequorea victoria. It is a naturally fluorescent protein which emits green light at a maximum wavelength of 509 nm when excited by blue or UV light. It is extensively used in laboratory as a reporter molecule to label and study cellular and subcellular proteins in living cells using a wide range of applications. Antibodies to GFP protein are used in immunoblotting and ELISA. GFP protein has molecular weight of 27 kDa.

Product citations

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