pAcGFP1-N In-Fusion Ready Vector encodes a green fluorescent protein from Aequorea coerulescens. The vector is prelinearized to enable directional In-Fusion cloning of PCR fragments onto the 5' end of the AcGFP1-encoding gene without further restriction digests. Sequences complementary to the vector sequences (flanking the site of linearization) need to be incorporated at the 5' ends of the PCR primers (see PT3865-2). The gene of interest is expressed as an N-terminal fusion to AcGFP1 in mammalian cells. The fluorescent protein coding sequence in this construct has been human codon-optimized for efficient expression and enhanced brightness in mammalian cells.

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AcGFP1 is a monomeric protein

AcGFP1 is a monomeric protein. Panel A. Recombinant AcGFP1 protein was analyzed by FPLC gel filtration chromatography. Overall protein absorbance (A₂₈₀) and chromophore excitation (A₄₇₇) of the eluted material were monitored simultaneously. AcGFP1 elutes from the column at a retention time corresponding to a molecular weight of 24 kDa. The calculated molecular weight of AcGFP1 is 26.9 kDa. Panel B. Recombinant AcGFP1 protein was analyzed by sucrose density ultracentrifugation using a continuous gradient. Panel C. Pseudonative gel analysis of proteins. The oligomeric structure of proteins is preserved during SDS-PAGE analysis if samples are kept at 4°C and not boiled prior to loading on a gel. Boiled and unboiled recombinant proteins (7.5 μg) were separated by SDS-PAGE electrophoresis (12% acrylamide). In both the boiled (denatured) and unboiled (nondenatured) samples, AcGFP1 runs as a uniform band of ~30 kDa due to its monomeric structure. The unboiled (nondenatured) DsRed-Express runs at a much higher molecular weight than its denatured (boiled) counterpart due to its oligomeric structure.

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Cloning of PCR-amplified alpha-actinin directly into four different In-Fusion Ready prelinearized DsRed-Monomer and AcGFP1 vectors

Cloning of PCR-amplified alpha-actinin directly into four different In-Fusion Ready prelinearized DsRed-Monomer and AcGFP1 vectors. The gene for alpha-actinin (1,600 bp) was amplified by PCR and immediately cloned into four different prelinearized DsRed-Monomer N1/C1 and AcGFP1 N1/C1 vectors using the In-Fusion cloning method. All four recombinant vectors were transfected into HeLa cells using a lipid-based transfection agent. 36 hr posttransfection, cells were fixed using 4% paraformaldehyde and visualized using a Zeiss Axioscope fluorescence microscope. Panel A. Alpha-Actinin-AcGFP1-C1. Panel B. Alpha-Actinin-AcGFP-N1. Panel C. Alpha-Actinin-DsRed-Monomer-C1. Panel D. Alpha-Actinin-DsRed-Monomer-N1.