The epitope for this antibody is in the peptide MVSKGAELFTGIVPILIE, the N-terminal 18 amino acids of the protein, which is found in most commercial GFP vectors and is distinct from the sequence seen in other fluorescent proteins. The homologous region of eGFP is MVSKGEELFTGVVPILVE, and this antibody binds this peptide also.
Supplied in Phosphate Buffered Saline with 50% Glycerol and 5mM Sodium Azide.
Storage
Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
General Notes
This antibody can be used to verify the expression, size, and stability of both AcGFP and eGFP fusion proteins in western blotting experiments, and to amplify GFP signals in tissues of transgenic animals.
Transfected HEK293 cells which overexpress GFP protein were stained with Anti-GFP Antibody. Cells which are transfected with GFP are bright green (left panel). Staining with Anti-GFP Antibody is shown in red (middle panel). The red and green signals overlap as expected (right panel). Most HEK293 cells are not transfected so only the nucleus of these cells can be visualized with a blue DNA stain.
Immunofluorescent analysis of HEK293 cells transfected with a GFP construct, in green, and stained with Anti-GFP Antibody [3B11] (A85303), at a dilution of 1:1,000, in red. The nuclear DNA is visualised in blue using Hoechst staining. The Anti-GFP Antibody [3B11] (A85303) binds to GFP protein expressed only in transfected cells, and as a result cells appear an orange-golden colour.
Figure 3: Western Blot - Anti-GFP Antibody [3B11] (A85303)
Western blot analysis of HEK293 cell lysates using Anti-GFP Antibody [3B11] (A85303), at a dilution of 1:1,000, in green. The lanes contain samples of: [1] Protein standards, [2] non-transfected control cells, [3] cells transfected with a GFP construct, and [4] cells transfected with an mCherry construct. The strong green band at approximately 27 kDa corresponds to GFP protein, detected only in cells transfected with GFP construct. The antibody does not recognise mCherry. The same blot was simultaneously probed with Anti-Heat Shock Protein 60 Antibody (A85438), at a dilution of 1:10,000, in red. The single band at 60 kDa represents HSP60 protein expressed in all preparations, as a positive loading control.
Figure 4: Western Blot - Anti-GFP Antibody [3B11] (A85303)
Crude homogenate from HEK293 cells transfected with pFin-EF1-GFP vector (Lane 1) and non-transfected HEK293 (Lane 2) was probed with Anti-GFP Antibody. The pFin-EF1-GFP vector expresses full length GFP. There is a strong clean band at about 27kDa corresponding to GFP which is absent from non-transfected cells.
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