Trypsin, DPBS, PBS, FBS, MEM, DMEM, penicillin/streptomycin, and l -glutamine were obtained from Thermo Fisher Scientific. Receptor ligands (5-HT, isoproterenol, ICI-118,551, and MT) and forskolin were purchased from Cayman Chemical or MilliporeSigma. Detergents (n-dodecyl-β-d -maltoside [DDM] and cholesteryl hemisuccinate [CHS]) were obtained from Anatrace. Digitonin, apyrase, GDPβS, and GDP were purchased from MilliporeSigma or BioBasic. [3H]SB269970 was obtained from PerkinElmer, and polyethylenimine (PEI) MAX was purchased from Polysciences.
N dodecyl β d maltoside ddm
Manufactured by Anatrace
Sourced in United States
N-dodecyl-β-D-maltoside (DDM) is a non-ionic detergent commonly used in biochemistry and cell biology. It is effective in solubilizing and stabilizing membrane proteins. DDM has a critical micelle concentration (CMC) of approximately 0.17 mM in water at 25°C.
Automatically generated - may contain errors
Lab products found in correlation
Cholesteryl hemisuccinate chs, by Anatrace (3 mentions)
Ni nta resin, by Qiagen (2 mentions)
Flag peptide dykdddd, by GenScript (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Benzonase, by Merck Group (2 mentions)
Isopropyl β d thiogalactopyranoside, by Merck Group (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Isoproterenol, by Cayman Chemical (1 mentions)
Polyethylenimine max pei, by Polysciences (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
3h sb 269970, by PerkinElmer (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Digitonin, by Merck Group (1 mentions)
Apyrase, by Merck Group (1 mentions)
Gdp β s, by Merck Group (1 mentions)
28 protocols using n dodecyl β d maltoside ddm
1
GPCR Ligand Binding Assay
2
Purification of BbZIP Protein Variants
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The expression of BbZIP was reported previously46 (link). In brief, the wild type or the variants of BbZIP was expressed in the strain of C41 (DE3) pLysS (Lucigen) in LBE-5052 Autoinduction medium for 24 h at room temperature. After harvest, spheroplasts were prepared and lysed in the buffer containing 20 mM Hepes (pH 7.3), 300 mM NaCl, 0.25 mM CdCl2, and cOmplete protease inhibitors (Sigma-Aldrich)83 (link). n-Dodecyl-β-D-maltoside (DDM, Anatrace) was added to solubilize the membrane fraction at the final concentration of 1.5% (w/v). The His-tagged protein was purified using HisPur Cobalt Resin (Thermo Fisher Scientific) in 20 mM Hepes (pH 7.3), 300 mM NaCl, 5% glycerol, 0.25 mM CdCl2, and 0.1% DDM. The sample was then concentrated and loaded onto a Superdex Increase 200 column (GE Healthcare) equilibrated with the gel filtration buffer containing 10 mM Hepes, pH 7.3, 300 mM NaCl, 5% glycerol, 0.25 mM CdCl2, and 0.05% DDM. For the variants with introduced cysteine residue(s), 1 mM TCEP was added during purification but excluded in the gel filtration buffer. The peak fractions were used for crystallization, cryo-EM, or crosslinking experiments.
3
Butterfly Flight Muscle Enzyme Characterization
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All chemicals were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) except n-dodecyl β-D-maltoside (DDM), obtained from Anatrace (Maumee, Ohio, USA). Butterflies were cooled in a refrigerator before dissection. Whole thoraces were dissected, weighed, flash-frozen in liquid nitrogen and stored at −80°C until biochemical analyses. To measure enzyme content and activity, the entire frozen thorax was sliced with a scalpel and immersed in 700–1000 µl of ice-cold homogenization buffer (50 mM potassium phosphate, pH 6.5 @ 22°C, 0.05% DDM); thorax tissue (more than 80% of which is flight muscle) was homogenized at 4°C in an ice-cold ground glass homogenizer. The insoluble pellet, composed primarily of chitin, was discarded after centrifugation for 10 min at 14,000 rpm, 4°C (5417R micro-centrifuge, Eppindorf Nordic, Copenhagen, Denmark). The volume of the resultant supernatant was measured, and homogenates were kept on ice until enzyme activities and concentrations were determined. CytOx activity of the pellet was assayed (details below) to ensure that mitochondrial membranes were solubilised. No residual enzyme activity was measured.
4
Purification of His-tagged BamABCDE Complex
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An E. coli bamABCDE construct containing all five genes with a C-terminal 8×His tag on BamE was expressed in E. coli BL21(DE3). At an OD600 of 0.6, the cells were induced with 0.5 mM isopropyl-β-d -1-thiogalactopyranoside (IPTG) and harvested after 4 h at 37°C. The cells were suspended in lysis buffer containing 25 mM Tris (pH 8.0), 300 mM NaCl, 5 mM imidazole, 10% glycerol, and 1× complete protease inhibitor mixture (Roche) and lysed with a Microfluidizer at 10,000 lb/in2. The cell lysate was supplemented with 1% n-dodecyl β-d -maltoside (DDM; Anatrace) and rocked overnight at 4°C. The suspension was ultracentrifuged at 125,000 × g for 1 h at 4°C. The supernatant was applied to a gravity flow column (Bio-Rad) packed with 5-ml preequilibrated nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen). The column was washed with five column volumes (CVs) of wash buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 50 mM imidazole, 10% glycerol, and 0.03% DDM and eluted with 5 CVs of elution buffer containing 300 mM imidazole. The eluent was applied to Superdex 200 16/60 column (GE Healthcare) that had been preequilibrated with the gel filtration buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl, and 1.5% n-octyl-β-d -glucopyranoside (OG; Anatrace).
5
Affinity Purification of Detergent-Solubilized Membrane Proteins
6
Purification and Mutagenesis of Human Ferroportin
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The cDNA of HsFpn (UniProt ID: Q9NP59) was codon optimized, synthesized, and cloned into a pFastBac dual vector. A tobacco etch virus (TEV) protease site and an octa-histidine (8×His) tag were added to the C-terminus of the protein. The Back-to-Bac method (Invitrogen) was used to express HsFpn was expressed in Sf9 (Spodoptera frugiperda). Purification of HsFpn follows the same protocol reported for TsFpn [2 (link)]. Size exclusion chromatography (SEC) was used to collect the purified HsFpn in FPLC buffer consisting of 20 mM HEPES (pH7.5), 150 mM NaCl, and 1 mM (w/v) n-dodecyl-β-D-maltoside (DDM, Anatrace). The Quikchange method (stratagene) was used to generate HsFpn mutations. Mutations were verified by sequencing. Mutant HsFpn proteins were expressed and purified following the same protocol for the WT.
7
Membrane Protein Purification via FLAG Affinity
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Cells were resuspended in 20 mM HEPES-Na pH 8.0, 300 mM NaCl, 1 mM TCEP, protease inhibitor cocktail (Sigma), and benzonase (Sigma). Cells were lysed by Dounce homogenizer and cellular debris were pelleted by low-speed centrifugation at 500 × g. Membranes were collected by centrifugation at 46,000 × g and stored at −80°C until use. Membranes were thawed and solubilized with the addition of 1% n-dodecyl β-D-maltoside (DDM) and 0.1% cholesteryl hemisuccinate (CHS) (10:1) (Anatrace). Debris and unsolubilized membranes were pelleted by centrifugation at 46,000 × g. The supernatant was subsequently used in FLAG affinity chromatography. The supernatant was applied to M1 anti-FLAG resin. The resin was washed with 20 bed volumes of 20 mM HEPES-Na pH 8.0, 300 mM NaCl, 1 mM TCEP, 0.005% lauryl maltose neopentyl glycol (LMNG), 0.0005% CHS (10:1) (Anatrace), and 5 mM ATP. The protein complex was eluted with the addition of 200 μg/mL of FLAG peptide (DYKDDDD) (GenScript). Protein was subsequently concentrated to >2 mg/mL and used for cryo-EM imaging.
8
Purification and Characterization of pMMO Membrane Protein
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pMMO was
purified as described
previously with some modifications.20 (link) The
membranes were isolated as described above, homogenized three times
using a Dounce homogenizer, and resuspended in lysis buffer at a final
concentration of 10–20 mg/mL, after which 1 mL aliquots were
flash-frozen in liquid N2 and stored at −80 °C.
Frozen membranes were thawed on ice and washed three times with 25
mM PIPES (pH 7.2), 0.5 M NaCl, 1 mM benzamidine, and 40 μM CuSO4. Washed membranes were then solubilized with the detergent n-dodecyl β-d -maltoside (DDM) (Anatrace)
by the addition of 1.5 mg of DDM/mg of protein at 4 °C for at
least 1 h while being gently stirred. The sample was concentrated
to 20 mg/mL using a Centriprep MWCO 50 device and loaded onto a Superdex
200 16/600 column (GE Healthcare) pre-equilibrated with 25 mM PIPES
(pH 7.2), 150 mM NaCl, 0.03% DDM, and 1 mM benzamidine. Sample purity
was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE), and pure fractions were collected and concentrated
with a Centriprep MWCO 50 device to 10–20 mg/mL. Samples were
flash-frozen and stored at −80 °C. The protein concentration
was determined by the Detergent-Compatible Lowry Assay (Bio-Rad) using
BSA as a standard. The recombinant spmoB protein, which corresponds
to the soluble domains of the pMMO pmoB subunit, was prepared as described
previously.12 (link),42 (link)
purified as described
previously with some modifications.20 (link) The
membranes were isolated as described above, homogenized three times
using a Dounce homogenizer, and resuspended in lysis buffer at a final
concentration of 10–20 mg/mL, after which 1 mL aliquots were
flash-frozen in liquid N2 and stored at −80 °C.
Frozen membranes were thawed on ice and washed three times with 25
mM PIPES (pH 7.2), 0.5 M NaCl, 1 mM benzamidine, and 40 μM CuSO4. Washed membranes were then solubilized with the detergent n-dodecyl β-
by the addition of 1.5 mg of DDM/mg of protein at 4 °C for at
least 1 h while being gently stirred. The sample was concentrated
to 20 mg/mL using a Centriprep MWCO 50 device and loaded onto a Superdex
200 16/600 column (GE Healthcare) pre-equilibrated with 25 mM PIPES
(pH 7.2), 150 mM NaCl, 0.03% DDM, and 1 mM benzamidine. Sample purity
was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE), and pure fractions were collected and concentrated
with a Centriprep MWCO 50 device to 10–20 mg/mL. Samples were
flash-frozen and stored at −80 °C. The protein concentration
was determined by the Detergent-Compatible Lowry Assay (Bio-Rad) using
BSA as a standard. The recombinant spmoB protein, which corresponds
to the soluble domains of the pMMO pmoB subunit, was prepared as described
previously.12 (link),42 (link)
9
Purification of Chimeric GLIC-5HT3A Receptors
10
Purification and Expression of FtsX Protein
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IU6892 (BL21AI pETCT‐ftsX‐GFP‐His) was grown in 50 mL LB broth to OD600 = 0.5, and temperature and inducer concentrations described in the text were used to express FtsX‐GFP‐His. After induction overnight, cells were lysed and FtsX‐GFP‐His expression was monitored by GFP fluorescence using a fluorescence plate reader (Spectramax 5 plate reader, Molecular Devices, CA; Ex = 395 nm; Em = 509 nm). Induced cells were collected by centrifugation at 4000g at 4°C for 10 min. Pellets from 1 L cultures were suspended in 35 mL lysis buffer (50 mmol/L Tris‐HCl pH 8.0, 200 mmol/L NaCl, 15% glycerol) and lysed by sonication (Branson digital sonifier; 1/8″ tapered microtip; pulse on = 0.5 sec/pulse off = 1.5 sec for 2 min; amplitude = 20%). Cell lysates were centrifuged in 1.5 mL polypropylene tubes at 100,000g at 4°C for 1 h to pellet membranes. Membranes were resuspended in lysis buffer additionally containing 1% (wt/vol) n‐dodecyl‐β‐d ‐maltoside (DDM) (Anatrace, Inc., OH. USA) for 2 h to solubilize membranes and centrifuged at 100,000g at 4°C for 30 min to pellet the insoluble material. The insoluble fraction was discarded and solubilized membranes were run on SDS‐PAGE. The position of FtsX‐GFP‐His on the SDS‐PAGE gel was determined by western blotting using anti‐GFP antibody. Different detergents were tested for optimal extraction of FtsX‐GFP‐His as described in the Results and Discussion .
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