The following method was adapted from Hong et al.19 (link) to determine the isomeric composition of the retinylidene chromophore of RGR in bovine RPE microsomes, before and after exposure to light. Samples were prepared and processed as for proteinase K digest, with the following changes. After protein precipitation and washing with MeOH and water, the protein pellet was resuspended in pronase-CHAPS buffer (100 mM BTP pH 7.8, 100 mM CaCl2, with 0.5% w/v CHAPS (Anatrace)). Subsequently, pronase (Roche) was added at approximately 10-times the weight of the RGR substrate. Then the digestion mixture was incubated at 8°C–10°C for 24 h with gentle agitation using a shaker. The digest was desalted using a BioPureSPN C18 spin column. The column was washed with 30% ACN to remove CHAPS, and the Nε-retinyl-Lys products were eluted with 50% ACN. The Nε-retinyl-Lys products in the eluent were separated using a Vanquish Flex HPLC system with an XBridge C18 column and a 16-min gradient of 30%–38% ACN in water with 0.1% FA at a flow rate of 0.3 mL/min. Nε-retinyl-Lys products were detected by HPLC absorbance at 330 nm and identified by MS/MS with CID fragmentation using the LTQ XL. The isomeric identities of the retinyl moieties bound to Lys were distinguished by their absorbance spectrum, using the 1260 Infinity HPLC system with the same chromatographic conditions.
Chaps
Manufactured by Anatrace
Sourced in United States
CHAPS is a zwitter-ionic detergent used for the solubilization and denaturation of proteins. It is effective for the extraction and purification of membrane proteins.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Pronase, by Anatrace (2 mentions)
Pronase, by Roche (2 mentions)
Ump glotm glycosyltransferase assay, by Promega (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Antarctic phosphatase, by New England Biolabs (2 mentions)
Non fat dry milk, by Bio-Rad (2 mentions)
Imagequant las 4000 system, by GE Healthcare (2 mentions)
Las 4000, by Cytiva (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Dithionite, by Thermo Fisher Scientific (1 mentions)
Alexa555 egf, by Thermo Fisher Scientific (1 mentions)
Protease arrest, by G Biosciences (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Nbd ps, by Avanti Polar Lipids (1 mentions)
Dna polymerase, by Thermo Fisher Scientific (1 mentions)
L α phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
1 2 dioleoyl sn glycero 3 phosphoserine, by Avanti Polar Lipids (1 mentions)
Luminata forte ecl detection system, by Merck Group (1 mentions)
23 protocols using chaps
1
Isomeric Analysis of RGR Chromophore
2
Isomeric Analysis of RGR Chromophore
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following method was adapted from Hong et al.19 (link) to determine the isomeric composition of the retinylidene chromophore of RGR in bovine RPE microsomes, before and after exposure to light. Samples were prepared and processed as for proteinase K digest, with the following changes. After protein precipitation and washing with MeOH and water, the protein pellet was resuspended in pronase-CHAPS buffer (100 mM BTP pH 7.8, 100 mM CaCl2, with 0.5% w/v CHAPS (Anatrace)). Subsequently, pronase (Roche) was added at approximately 10-times the weight of the RGR substrate. Then the digestion mixture was incubated at 8°C–10°C for 24 h with gentle agitation using a shaker. The digest was desalted using a BioPureSPN C18 spin column. The column was washed with 30% ACN to remove CHAPS, and the Nε-retinyl-Lys products were eluted with 50% ACN. The Nε-retinyl-Lys products in the eluent were separated using a Vanquish Flex HPLC system with an XBridge C18 column and a 16-min gradient of 30%–38% ACN in water with 0.1% FA at a flow rate of 0.3 mL/min. Nε-retinyl-Lys products were detected by HPLC absorbance at 330 nm and identified by MS/MS with CID fragmentation using the LTQ XL. The isomeric identities of the retinyl moieties bound to Lys were distinguished by their absorbance spectrum, using the 1260 Infinity HPLC system with the same chromatographic conditions.
3
UMP-Glo Glycosyltransferase Assay for MraY
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The UMP-GloTM glycosyltransferase assay64 (link) was carried out according to the manufacturer′s specifications (Promega Corporation). For both IC50 and specific activity measurements, reaction mixtures contained 150 μM UDP-MurNAc-pentapeptide (UM5A) and 250 μM undecaprenyl phosphate (C55-P) in a buffer composed of 100 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl2, and 20 mM (3-((3-cholamidopropyl) dimethylammonio)−1-propanesulfonate) (CHAPS; Anatrace). The reaction was initiated with MraYAA to a final concentration of 50 nM. Reactions were carried out for 5 min at 45 °C. All luminescence measurements were normalized relative to a negative control reaction without enzyme. For IC50 measurements, the following concentrations were used. Carbacaprazamycin: 0.01, 0.05, 0.1, 0.5, 1, 20, and 220 μM; capuramycin: 0.01, 0.1, 0.5, 1, 2.5, 50, and 375 μM; 3′-hydroxymureidomycin A: 0.01, 0.05, 0.1, 1, 5, 50, and 370 μM. For specific activity measurements, NB7 and each inhibitor were added to a final concentration of 1 μM and 0.5 μM where present. Luminescence measurements were made using a SpectraMax M3 multi-mode microplate reader.
4
Isolation and Solubilization of B. subtilis Membrane Proteins
5
Characterization of Lipid Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following reagents were purchased from the manufacturers as noted: cholesterol (C8503), sulfatide, PA, ethanolamine, ATP and n-octyl-β-D-glucopyranoside (Sigma); and PI (Echelon); di16:0-PIPs (CellSignals); PS, PE, and PC (porcine brain) for liposome cosedimentation assay, 1,2-dioleoyl-PC, 1,2-dioleoyl-PE, 1,2-dioleoyl-PS, 1,2-dioleoyl-PI, 1,2-dioleoyl-PG, 1,2-dioleoyl-PA, brain polar lipids (porcine), sphingomyelin (porcine brain), cholesterol (ovine wool), C6-NBD-PS and C6-NBD-PE (Avanti Polar Lipids); dithionite (Fisher); CHAPS (Anatrace); and Alexa 594-Tfn, Alexa 594-CTxB, Alexa 555-EGF (Invitrogen). To prepare Alexa 488-Tfn, human holo-Tfn (Sigma) was conjugated with Alexa 488 using Alexa Flour succinimidyl ester (Invitrogen) and then purified by PD-10 desalting columns (GE Healthcare).
6
In vitro liposome reconstitution of FtsZ and FtsA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
50 μl of E. coli total lipid extract chloroform solution at 10
mg/ml was dried in a glass vial under a stream of nitrogen gas and left overnight
under vacuum to remove traces of the solvent. The resulting thin lipid film was
hydrated with 50 μl of TEN1007.5 plus 20 mM CHAPS (Anatrace, Maumee,
Ohio) and shaken vigorously at 800 rpm using a benchtop Eppendorf shaker for 2 hr.
The lipid–detergent solution was sonicated for 1 min in a water bath
sonicator. Subsequently, 50 μl of TmFtsZ (30 µM) and TmFtsA (10 µM)
solutions supplemented with 0.5 mM MgGTP or MgGMPCPP (Jena Bioscience, Germany) or no
nucleotide was added and left for 30 min at room temperature. Next, the mixture was
gradually diluted within 10 to 20 min to 600 μl with TEN1007.5 or
TEN1007.5 plus nucleotides (both without detergent) to trigger
spontaneous liposome formation. 2.5 µl of the solution was mixed with 0.2
µl 5 nm IgG immunogold conjugate (TAAB, UK) and plunge-frozen onto Quantifoil
R2/2 holey carbon grid using an FEI Vitrobot.
mg/ml was dried in a glass vial under a stream of nitrogen gas and left overnight
under vacuum to remove traces of the solvent. The resulting thin lipid film was
hydrated with 50 μl of TEN1007.5 plus 20 mM CHAPS (Anatrace, Maumee,
Ohio) and shaken vigorously at 800 rpm using a benchtop Eppendorf shaker for 2 hr.
The lipid–detergent solution was sonicated for 1 min in a water bath
sonicator. Subsequently, 50 μl of TmFtsZ (30 µM) and TmFtsA (10 µM)
solutions supplemented with 0.5 mM MgGTP or MgGMPCPP (Jena Bioscience, Germany) or no
nucleotide was added and left for 30 min at room temperature. Next, the mixture was
gradually diluted within 10 to 20 min to 600 μl with TEN1007.5 or
TEN1007.5 plus nucleotides (both without detergent) to trigger
spontaneous liposome formation. 2.5 µl of the solution was mixed with 0.2
µl 5 nm IgG immunogold conjugate (TAAB, UK) and plunge-frozen onto Quantifoil
R2/2 holey carbon grid using an FEI Vitrobot.
7
Characterization of Phospholipid Reagents
Check if the same lab product or an alternative is used in the 5 most similar protocols
l -α-Phosphatidylcholine (egg, chicken; ePC), DOPC, 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), PI4P, PI, and NBD-PS were purchased from Avanti Polar lipids (Alabaster, AL). ATP, adenylyl-imidodiphosphate (AMP-PNP), and octyl-β-d -glucopyranose (OGP) were purchased from Sigma-Aldrich, sodium hydrosulfite (sodium dithionite) from Fischer Scientific, CHAPS from Anatrace (Maumee, OH), Protease Arrest from G-Biosciences (St. Louis, MO), and 1D4 peptide from Celtek Peptides (Franklin, TN). HEK293T and rat PC12 cells were purchased from ATCC. Rho 1D4 antibody used for preparation of immunoaffinity columns was generated in-house (Hodges et al., 1988 (link); Quazi and Molday, 2013 (link)) and purchased from UBC through Flintbox (www.rho1d4.com/ ); primary antibodies against calnexin, actin, and tubulin were from Abcam; fluorescent-tagged secondary antibodies for immunofluorescence imaging were from Molecular Probes; and anti-Cdc50-7F4 (CDC50A) primary antibodies used in Western blots and immunofluorescence analysis were raised in-house (Coleman and Molday, 2011 (link)). Restriction enzymes, T4-DNA ligase, Antarctic Phosphatase, and CaMKII kit were procured from New England Biolabs, DNA polymerase was from Thermo Scientific, and the QuikChange Site-Directed Mutagenesis Kit was from Agilent Technologies.
+ Expand
8
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with phosphate-buffered saline (PBS) containing 20 mM N-ethylmaleimide (NEM) for 1 min and then lysed with 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Anatrace) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–buffered saline, pH 6.8, supplemented with 20 mM NEM and protease inhibitors for 20 min on ice. Postnuclear supernatants (PNSs) were collected by centrifugation at 10,000 × g for 10 min. Samples were denatured and reduced in dithiothreitol (DTT)-containing sample buffer for 10 min at 65°C and separated by SDS–PAGE. Proteins were transferred to PVDF membranes with the Trans-Blot Turbo Transfer System (Bio-Rad, Cressier, Switzerland). Membranes were blocked with 10% (wt/vol) nonfat dry milk (Bio-Rad) and stained with the aforementioned primary antibodies and horseradish peroxidase–conjugated secondary antibodies. Membranes were developed using the Luminata Forte ECL detection system (Millipore, Schaffhausen, Switzerland), signals were detected with the ImageQuant LAS 4000 system in the standard acquisition mode (GE Healthcare Life Sciences, Glattbrugg, Switzerland), and bands were quantified using the Multi Gauge Analysis tool (Fujifilm). For each antigen, the linearity of the detected signal range was ensured with appropriate loading controls.
9
Lipid Reconstitution Protocol Optimization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
10
Cryo-EM Sample Preparation for Nanodiscs and Protein Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cryo-EM samples were prepared using a CP3 cryoplunge (Gatan). Quantifoil R0.6/1 Cu300 holey carbon grids were cleaned with chloroform, acetone, and isopropanol as described by Passmore and Russo, 2016 (link). The grids were glow discharged in the ELMO glow discharge system (Corduan Technologies) from both sides for 2 min at 11 mA and 0.28 mbar. For the nanodisc-reconstituted sample, four microliters of the reconstituted protein solution at 0.15 mg ml−1 concentration were applied on a grid and blotted from both sides for 2.2 s with Whatman No. 3 filter paper at 97% relative humidity. The LMNG-purified complex I was supplemented with 0.2% CHAPS (Anatrace) and applied at concentration 2–3 mg ml−1. The grids were plunge-frozen in liquid ethane at −176°C and stored in liquid nitrogen.
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!