GFP | Green Fluorescence Protein (affinity purified)

PRODUCT INFORMATION IN PDF

product information 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. Immunogen highly purified native GFP protein derived from Aequorea victoria, UniProt: P42212 Host Rabbit Clonality Polyclonal Clone Purity Affinity purified serum Format Liquid Quantity 100 µg Reconstitution Storage Store at 4°C, or in small aliquots at -20°C. Avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tubes. Tested applications ELISA (ELISA), Immunofluorescence (IF), Western blot (WB) Related products AS15 3000 | GFP (protein A purified), rabbit antibody AS15 2999 | GFP (total IgG), rabbit antibody AS15 3001 | GFP, peroxidase conjugated, rabbit antibody AS15 2998 | GFP (total IgY), chicken antibody AS15 3002 | GFP (purified recombinant protein) other tag antibodies Additional information

application information
Recommended dilution		1 : 5000-1 : 25 000 (ELISA), 1 : 500 (IF), 1 : 2000-1 : 10 000 (WB)
Expected | apparent MW
Confirmed reactivity		Native GFP, Recombinant GFP (E.coli), all variants of GFP
Predicted reactivity
Not reactive in
Additional information		Minimal cross-reactivity with E.coli proteins.
Selected references		Kulich et al. (2018). Deubiquitinase OTU5 affects Root Responses to Phosphate Starvation. Plant Physiol. 2018 Jan 4. pii: pp.01693.2017. doi: 10.1104/pp.17.01693.

Application example

immunolocalization



Detection of YFP-tagged Lamin A in the nucleus of mouse fibroblasts using GFP | Green Fluorescence Protein (affinity purified) antibodies. Fixation and permeabilization was performed with methanol at -20°C for 10 min. a) Indirect immunofluorescence labeling of YFP-lamin A with anti-GFP primary antibody (dilution 1: 500) as primary antibody and detected by TRITC-conjugated goat-anti-rabbit as secondary antibody.
b) Green fluorescence image of YFP-tagged Lamin A, which is incorporated into the nuclear lamina and tubular structures that penetrate into the nucleus.
c) DAPI staining of nuclear DNA.
d) Merged images

western blot 



Protein extracts were obtained from Arabidopsis thaliana seeds (producing atLEA4-5 fused to GFP; other lanes contain GFP-6H loaded in indicated amounts). Following extraction buffer was used: 0.7 M sucrose, 0.5 M Tris-base, 30 mM HCl, 50 mM EDTA, 0.1 M KCl, 2% β-mercaptoethanol, 12 mg/ml poly-vinyl-poly-pyrrolidone (PVPP). This buffer was complemented with 2mL of equilbrated phenol before extraction. Samples were centrifuged and the protein phase was recovered; the extracted proteins were precipitated with 0.1 m ammonium acetate dissolved in methanol, centrifuged and the pellet washed with cold (-20 ℃) 80% acetone. The protein pellet was dissolved in SDS-solubilization buffer (1% CHES, 2% SDS, 2% ß-mercaptoethanol, 10% glicerol). Thirty 𝜇g of seed protein extract was denatured with Laemmli buffer at 95 °C for 5 min and proteins were separated on 15% SDS-PAGE and blotted 1.5 h to nitrocellulose membrane in a liquid transfer system. Blots were blocked with 2% skim milk ON at 4°C with agitation. After rinsing with TBS, blots were incubated in the primary antibody at a dilution of 1:10 000 (anti-GFP) for 3h at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed once for 15 min. and 3 times for 5 min in TBS-T at 4°C with agitation. Then, blots were incubated in secondary antibody (anti-rabbit IgG horse-radish peroxidase conjugate) diluted to 1:25 000 in for 1h at RT with agitation. The blot was washed as above and developed for 2 min with SuperSignal West Pico Chemiluminescent Substrate, Thermo Fisher Scientific. Exposure time was for 30 seconds.

Courtesy of Dr. Alejandra Covarrubias, UNAM, Mexico

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