Restriction enzymes (BamHI and EcoRI) and T4 ligase were purchased from Sangon Biotech Co., Ltd. Rabbit polyclonal or monoclonal antibodies against human cytokeratin 7 (cat. no. ab181598), Bax (cat. no. ab32503), Bcl-2 (cat. no. ab196495), PI3K (cat. no. ab86714), optineurin (OPTN; cat. no. ab151240), Akt1/2/3 (cat. no. ab184136), phosphorylated (p)-Akt1 (T308; cat. no. ab105731), excision repair cross-complementation group 1 (ERCC1; cat. no. ab129267) and β-actin (cat. no. ab8227) were purchased from Abcam. Rabbit polyclonal antibodies against human caspase-3 p17 (cat. no. sc-271,028) and EEF1D (cat. no. abs136494) were purchased from Santa Cruz Biotechnology, Inc., and Absin Bioscience, Inc., respectively. Lipofectamine® 3000 transfection reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The HRP-conjugated secondary antibody (goat anti-mouse or goat anti-rabbit IgG, cat. no. PV-6000) were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. FITC-labeled goat anti-rabbit IgG (H + L) secondary antibody (cat. no. A0562) and Hoechst 33342 (cat. no. C1022) were purchased from Beyotime Institute of Biotechnology. LY294002 (PI3K inhibitor; cat. no. ab120243) and MK-2206 (Akt inhibitor; cat. no. SF2712) were purchased from Abcam and Beyotime Institute of Biotechnology, respectively.
Bamh 1
Manufactured by Sangon
Sourced in China
BamH I is a type II restriction endonuclease enzyme that recognizes and cleaves the palindromic DNA sequence 5'-GǴATCC-3'. It is commonly used in molecular biology applications for DNA manipulation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ecori, by Sangon (4 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Sangon (3 mentions)
Testosterone, by Sangon (3 mentions)
Hind 3, by Sangon (3 mentions)
Nde 1, by Sangon (3 mentions)
Dna marker and protein marker, by Thermo Fisher Scientific (3 mentions)
T4 ligase, by Sangon (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Ecori, by Takara Bio (1 mentions)
Puc57 vector, by Sangon (1 mentions)
Bamhi, by Takara Bio (1 mentions)
Xhoi, by Sangon (1 mentions)
E coli top10, by Sangon (1 mentions)
E coli bl21 de3, by Sangon (1 mentions)
Plasmid mini prep kit, by Sangon (1 mentions)
Pgex 4t 1, by Sangon (1 mentions)
7 protocols using bamh 1
1
Antibody and Reagents for Cell Signaling
2
Cloning and Expression of CiSA32.6t Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PUC57 vector with the truncated CiSA32.6 (CiSA32.6t) gene (852 bp) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. A set of oligonucleotides, 5′-TAGGATCCGAGAAAAAACTGCAGTTG-3′ and 5′-GCGAATTCTCAAGTAGAGGTAACTTC-3′ (the underlined letters indicate BamH I and EcoR I recognition sites, respectively) and the recombinant plasmid pUC57/CiSA32.6 were used respectively as the primers and the template DNA to amplify the CiSA32.6t by PCR. The purified PCR product and pET28a were doubly digested by BamH I and EcoR I (TAKARA, Dalian, China), respectively. Then, the two products were ligated together and transformed into Escherichia coli BL21.
E. coli containing plasmid pET28a/CiSA32.6t was cultured in liquid LB medium at 37℃ until OD600nm reached between 0.3 and 0.5. E. coli was induced to express rCiSA32.6t by the addition of isopropylthio-β-D-galactoside (IPTG) (Sangon, Shanghai, China) and incubation for another 6 h at 25℃. rCiSA32.6t was purified by metal affinity chromatography using Ni+2-NTA Sepharose (Sangon) according to the instructions from the manufacturer. The purified protein was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis.
E. coli containing plasmid pET28a/CiSA32.6t was cultured in liquid LB medium at 37℃ until OD600nm reached between 0.3 and 0.5. E. coli was induced to express rCiSA32.6t by the addition of isopropylthio-β-D-galactoside (IPTG) (Sangon, Shanghai, China) and incubation for another 6 h at 25℃. rCiSA32.6t was purified by metal affinity chromatography using Ni+2-NTA Sepharose (Sangon) according to the instructions from the manufacturer. The purified protein was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis.
3
Biodegradation of NOD by Recombinant Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant pGEX-4T-1/USTB-05-A/BL21(DE3) and pET30a(+)/USTB-05-C/BL21(DE3) were previously studied on MCs and used in this study for NOD biodegradation [30 (link),49 (link)]. E. coli TOP10 and E. coli BL21(DE3) were purchased from Sangon Biotech (Shanghai, China). The recombinant and host strains were cultured in Luria–Bertani (LB) medium [41 (link)] on a shaker at 200 rpm at an appropriate temperature. The vectors pGEX-4T-1 and pET30a(+), restriction enzymes BamHI, XhoI, SacI and NotI, the plasmid mini-prep kit and the polymerase chain reaction (PCR) kit were obtained from Sangon Biotech (Shanghai, China). Standard NOD was purchased from Enzo Science Inc., Farmingdale, NY, USA. All other chemicals were analytical grade.
4
Nucleolin Knockdown by siRNA Lentivirus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four siRNA oligonucleotides targeting the mRNA of nucleolin (NCL-siRNA), as well as a scrambled RNA oligonucleotide, were designed and synthesized with BamHI and EcoRI restriction site enzymes (Sangon Biotech Co., Ltd.; Shanghai, China). The two siRNAs with the highest interference performance were used in subsequent studies (NCL- siRNA1: gatccGCGATGAGGATGACGAAGATTTCAAGAGAATCTTCGTCATCCTC ATCGTTTTTT, NCL-siRNA4: gatccGGAAGACGGTGAAATTGATTTCAAGAGAATCAATTTCACCGTCTTCC TTTTTT). The siRNAs and scrambled RNA oligonucleotides were cloned into pLVX-shRNA1 plasmids. The pseudotyped lentiviruses were produced by co-transfecting recombinant siRNA plasmids with Clontech Lenti-X HT packaging vectors into 293T cells. After 48 h, the lentivirus-containing supernatants were collected by centrifugation at 1,200 rpm for 10 min. L-02 cells were treated with supernatants for 24 h and stable nucleolin knockdown cells were isolated after 2 weeks of selection with puromycin (2 μg/mL). Knockdown efficiency was verified by Western blot analysis. SET-siRNA production and RNA interference efficacy have been described and validated in our previous studies (Liu et al. 2007; Yang et al. 2012).
5
Synthesis and Characterization of AuNPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gold nanoparticles (AuNPs) was kindly provided by Prof Zhenxin Wang (National Analytical Research Center of Electrochemistry and Spectroscopy, Changchun, China).
Hind III, EcoR I, Nde I, BamH I, testosterone, and IPTG were purchased from Sangon Biotech (Shanghai, China). DNA marker and protein marker were purchased from Thermo (USA).
Hind III, EcoR I, Nde I, BamH I, testosterone, and IPTG were purchased from Sangon Biotech (Shanghai, China). DNA marker and protein marker were purchased from Thermo (USA).
6
Synthesis and Characterization of AuNPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gold nanoparticles (AuNPs) was kindly provided by Prof Zhenxin Wang (National Analytical Research Center of Electrochemistry and Spectroscopy, Changchun, China).
Hind III, EcoR I, Nde I, BamH I, testosterone, and IPTG were purchased from Sangon Biotech (Shanghai, China). DNA marker and protein marker were purchased from Thermo (USA).
Hind III, EcoR I, Nde I, BamH I, testosterone, and IPTG were purchased from Sangon Biotech (Shanghai, China). DNA marker and protein marker were purchased from Thermo (USA).
7
Synthesis and Characterization of AuNPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gold nanoparticles (AuNPs) was kindly provided by Prof Zhenxin Wang (National Analytical Research Center of Electrochemistry and Spectroscopy, Changchun, China).
Hind III, EcoR I, Nde I, BamH I, testosterone, and IPTG were purchased from Sangon Biotech (Shanghai, China). DNA marker and protein marker were purchased from Thermo (USA).
Hind III, EcoR I, Nde I, BamH I, testosterone, and IPTG were purchased from Sangon Biotech (Shanghai, China). DNA marker and protein marker were purchased from Thermo (USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!