AKAP7γ-pET32a, AKAP7α-EGFP-N1, and AKAP7γ-EGFP-N1 were used as previously described [22 (link), 25 (link), 27 (link)]. AKAP7γ 1-150-EGFP, AKAP7γ 150-end-EGFP, and AKAP7γ-1-268-pET32a were constructed by PCR amplification and subcloning into the EcoRI-BamHI sites of pEGFP-N1 (Clontech) or the EcoRI-HindIII sites of pET32a (Millipore). AKAP7γ-pMAL was constructed by PCR amplification and subcloning into the BamHI-EcoRI sites of pMal-c5E (NEB). AKAP7γ-mCherry was constructed by PCR amplification and subcloning into the EcoRI-BamHI sites of pmCherry-C1 (Clontech). AKAP7γ-ΔPKA-EGFP was made by site directed mutagenesis by Genewiz. Monomeric EGFP and tandem EGFP used for PCH analysis were obtained from the lab of Dr. Kathy Herrick-Davis. The AKAP18δ-YFP construct was kindly provided by Prof. Enno Klussmann.
Pmal c5e
Manufactured by New England Biolabs
Sourced in United States
PMal-c5E is a laboratory equipment product used for protein purification. It functions as an affinity chromatography matrix that selectively binds to maltose-binding protein (MBP) tagged proteins. This allows for the efficient isolation and purification of MBP-tagged recombinant proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Pmcherry c1, by Takara Bio (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Ampicillin sodium salt, by Merck Group (1 mentions)
Plasmid miniprep kit, by Merck Group (1 mentions)
Dpni enzyme, by Thermo Fisher Scientific (1 mentions)
Escherichia coli dh5α, by New England Biolabs (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Luria bertani broth, by HiMedia (1 mentions)
Gibson assembly cloning kit, by New England Biolabs (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
4 protocols using pmal c5e
1
Recombinant AKAP7 Protein Constructs
2
Bacterial Expression System for Mutagenesis
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Escherichia coli DH5α (New England Biolabs (NEB), Ipswich, MA, U.S.A.), pMAL c5E (NEB), Plasmid miniprep kit (Sigma chemicals, St. Louis, MO, U.S.A.), Quick-change site-directed mutagenesis kit (Stratagene, Cedar Creek, TX, U.S.A.), Ampicillin sodium salt (Sigma), Primers (Sigma), DpnI enzyme (Fermentas, Waltham, Massachusetts, United States), IPTG (Sigma), Luria-Bertani (LB) broth (Himedia, Mumbai, India).
3
Cloning and Mutagenesis of PAR-1 and MEX-5 Constructs
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Table S3 lists all plasmids used in this study. A Q5 site-directed mutagenesis kit (New England Biolabs) was used to integrate the 6xHis coding sequence at the N-terminus of the maltose-binding protein (MBP) coding sequence in vector pMAL-C5E (New England Biolabs), to generate pAF9. The 6xHis::MBP::PAR-1 and 6xHis::MBP::PAR-1(ΔKA1) expression vectors were constructed by cloning par-1 and par-1(ΔKA1) open reading frames (ORFs) into pAF9. The 6xHis::MBP::MEX-5(452-460) expression vector was constructed by cloning mex-5 ORFs into pAF9 using a Gibson assembly cloning kit (New England Biolabs). The 6xHis::KA1 expression vector (pAF10) was constructed by cloning par-1(1089-1192) ORF into pET28a (Novagen) using a Gibson assembly cloning kit (New England Biolabs). A Q5 site-directed mutagenesis kit (New England Biolabs) was used to generate the 6xHis::KA1(KRSS) expression vector (pAF11) (Tables S3,S4 ).
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4
Recombinant Expression of Hexokinase G6PDHs
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To identify the enzyme activity of target proteins, recombinant vectors expressing HbG6PDHs were constructed to prokaryotic expression in E. coli. Four HbG6PDH ORFs were amplified using primers with different restrict enzyme sites (Table 1 ), and then inserted into the plasmid pMAL-c5E (New England BioLabs) in the same translational frame as the malE gene that encodes a maltose-binding protein (MBP). The resulting recombinant vectors were transformed into the E. coli BL21 (DE3) to generate the recombinant proteins. The transformed clones were cultivated at 37°C in liquid LB medium supplemented with 100 μg/ml ampicillin and 25 μg/ml chloramphenicol. The isopropyl β-D -1-thiogalactopyranoside (IPTG) was added at 0.4–0.6 of OD600nm to a final concentration of 1.0 mM. After shaking at 30°C for 2 h, the induced strains were broken by ultrasonic wave with 200 w for 5 min, and then centrifuged at 10,000g for 10 mins. The supernatant was collected and stored for protein electrophoresis detection and enzyme activity assay.
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