The full-length cDNA for human MYH11 (SM1A isoform) was truncated at the codon for threonine 1775 [thus generating the heavy meromyosin (HMM) fragment], after which a glycine plus FLAG peptide (DYKDDDDK) was appended to facilitate purification. Site-directed mutagenesis was performed using QuikChange XLII kit (Stratagene) to introduce the S237Y mutation into the same construct. The constructs were subcloned into the baculovirus transfer vector p2Bac (Invitrogen). Protein expression and purification were performed as previously described (Sweeney et al., 1998 (link)). The HMM construct had been previously subcloned into the baculovirus transfer vector pVL 1393 (Invitrogen). Baculovirus expression was used to produce HMM fragments of smooth muscle myosin after infection of an insect cell line (Sf9) with recombinant baculovirus (Sweeney et al., 1998 (link)). Mutagenesis of full-length MYH11 containing the corresponding L1287M amino acid substitution (V1289M in human SMA1 MYH11 isoform) was performed by site-directed mutagenesis by Mutagenex (Hillsborough, NJ).
Pvl1393
Manufactured by Thermo Fisher Scientific
The PVL1393 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for use in scientific research and analysis applications. The core function of the PVL1393 is to provide a controlled environment for specific laboratory processes.
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Lab products found in correlation
5 protocols using pvl1393
1
Smooth Muscle Myosin Mutant Expression
2
Cloning and Expression of JAML Constructs
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All plasmid constructs were generated by the Custom Cloning Center Facility at Emory University and sequences confirmed. Human JAML (GenBank: AJ515553.2) was cloned in pcDNA3.0 (Invitrogen Life Technologies) with C-terminal His-tags or 6XMyc or rabbit-Fc 20 (link). The extracellular domains of JAML were tagged after residue Leu259 for JAML.D1D2-His (sJAML), and Pro140 for sJAML.D1. CHO or HEK293 T cells were transfected using polyethylenimine (PEI), and stable clones selected with G418/ HygromycinB appropriately. Soluble His-tagged proteins were purified on Ni-NTA agarose beads (Qiagen, Valencia, CA). Soluble CAR-GST construct has been described9 (link). Full-length CAR and JAML constructs were expressed in CHO cells (CHO-CAR or CHO-JAML) and expression assessed by flow cytometry. Adenofiber knob proteins Ad5-His (Ad5) and Ad11-His (Ad11) were expressed in E.Coli from respective constructs (a kind gift of Dr. G. Nemerow, Scripps Institute, LaJolla,CA). Soluble murine JAML–mFc construct was cloned into pVL1393 (Invitrogen), expressed in the Baculovirus system (Invitrogen) and purified on Protein A Sepharose.
3
Cloning and Expression of JAML Constructs
4
Baculoviral Expression of Porcine p110γ-p84 Complex
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Constructs of full-length porcine p110γ were cloned into pVL1393 (Invitrogen). The plasmid for EE-tagged mouse p84 was a gift from Len Stephens (The Babraham Institute, UK). The constructs were transfected into Spodoptera frugiperda 9 (Sf9) insect cells with ExGen500 (Fermentas) and incubated at 27°C for 5 days to make baculoviruses. The heterodimeric p110γ-p84 complexes were obtained by co-infection of 3 L of SF9 cells with p110γ-expressing and p84-expressing viruses. Cells were inoculated at a density of 1 × 107 (link) cells/mL and grown in 2L roller bottles standing vertically, with 500 mL of Sf9 cells per bottle. After 62 h incubation at 27°C, cells were harvested, washed in PBS, pelleted, snap-frozen in liquid nitrogen and stored at −80°C.
5
Recombinant Expression and Purification of Hybrid SALM5 Protein
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The Ig1-3-FNIII12 domain (aa 27A–513A), Ig1-3-A+B+ (aa 30D-318L), and FN12 (aa 312Q–510G) of human LAR were cloned into pAcGP67 (BD Bioscience), modified for C-terminal protein-A tagging. Since human SALM5 was hardly expressed in High Five insect cells (Invitrogen), hybrid LRR techniques were used to generate a chimeric hySALM5 proteins67 (link). Briefly, the N terminus of VLRB61 (1M–82L) was combined with the LRR and Ig domains of human SALM5 (aa 59A–374I) by overlap PCR, and the resulting chimeric gene for hySALM5 was cloned into pVL1393 (Invitrogen), modified for C-terminal Fc tagging. All constructs described above contain a thrombin cleavage site (LVPRGS) between target proteins and tags. The High Five insect cells were transfected with corresponding P4 baculovirus for 3 days and harvested. The supernatants containing secreted proteins were loaded onto IgG sepharose column to purify protein A-fused proteins, or onto protein A sepharose column to purify Fc-tagged protein. The protein A or Fc tags were thrombin cleaved (0.5% v/v) for 16 hours at 4 °C, followed by gel-filtration purification using Superdex 200 (GE Healthcate Life Science).
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