All reagents, unless otherwise stated, were purchased from Sigma Aldrich. Nickel-NTA agarose resin and isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from Gold Biotechnology. Sodium dodecyl sulfate (SDS), acrylamide, and tetramethylethylenediamine (TEMED) were purchased from Bio-Rad and Luria-Broth powder was purchased from IBI Scientific. During the protein expression process, cells and cell lysate were pelleted using an Avanti J-E centrifuge (Beckman Coulter). Proteins were concentrated using an Allegra X-14R centrifuge with three types of 3-kDa molecular weight cutoff (MWCO) centrifugal units: Amicon Ultra (Millipore, 15 mL capacity), Vivaspin (Sartorius Stedim Biotech, 2 mL capacity), and Vivaspin (Sartorius Stedim Biotech, 0.5 mL capacity). The Ec-ACP plasmid pTL1426 (link) (N- and C-terminal His6 tags; kanamycin resistant) was provided by the Khosla Lab at Stanford University. The Ec-ACP plasmid pKJ5535 (N-terminal His6 tag only; kanamycin resistant) was constructed as described in Supporting Information . The ACPact plasmid (pMC002067; carbenicillin resistant) was provided by the Chang Lab at University of California Berkeley.
X 14r centrifuge
Manufactured by Merck Group
The X-14R centrifuge is a compact and versatile laboratory equipment designed for a wide range of applications. It features a maximum speed of 14,000 rpm and a maximum relative centrifugal force of 20,000 x g, making it suitable for a variety of sample separation and preparation tasks. The centrifuge is equipped with a motorized lid and an easy-to-use control panel for precise speed and time settings.
Automatically generated - may contain errors
Lab products found in correlation
Avanti j e centrifuge, by Beckman Coulter (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by GoldBio (1 mentions)
Tetramethylethylenediamine, by Bio-Rad (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Vivaspin, by Sartorius (1 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
2 protocols using x 14r centrifuge
1
Recombinant Protein Expression and Purification
2
Biotin Conjugation of Protein Samples
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Reactive EZ-link sulfo-NHS-LC-biotin (Thermo Fisher Scientific) was dissolved in 1× PBS pH 7.4 to obtain a final concentration of 10 mM. Protein conjugates containing biotin were prepared by incubating SA fusion proteins (at concentrations of 2 mg mL−1 in 1× PBS pH 7.4) with ten-fold molar excess of EZ-link sulfo-NHS-LC-biotin for 30 min at room temperature. Excess of unreacted or hydrolyzed biotinylation reagent was removed using SEC with Superdex 200 10/300 GL connected to an AKTApurifier system and equilibrated with buffer 1× PBS pH 7.4. Fractions corresponded to the expected protein pick were pulled and concentrated to a final concentration of 2 mg mL−1 using 10000 NMWL Amicon Ultra-4 ultrafiltration devices (Millipore) at 4000 × g and 4 °C on a Allegra X-14R centrifuge. Final protein concentrations were measured using a NanoDrop 2000 Spectrophotometer.
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