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Caps buffer

Manufactured by Merck Group

CAPS buffer is a commonly used buffer solution in molecular biology and biochemistry laboratories. It is a zwitterionic buffer with a pKa of approximately 10.4 at 25°C. CAPS buffer is effective for maintaining pH in the alkaline range, typically between pH 9-11, and is often used in various applications such as protein purification, enzyme assays, and electrophoresis.

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7 protocols using caps buffer

1

LAMP-2 Isoforms Sensitivity to Sunitinib

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Cells overexpressing LAMP-2A, LAMP-2B, and LAMP-2C were harvested and seeded into a 96-well plate (5×103 cells/well), and sunitinib was added at various concentrations (2.5, 5.0, 10, 20, 40 and 80 µM). The plates were incubated for 72 h, and the cells were fixed with 5% glutaraldehyde for 30 min at room temperature and then stained with 0.2% crystal violet with CAPS buffer (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. The crystal violet staining was dissolved with 10% acetic acid and quantified using a microplate reader at 495 nm. The results of the assay were the average value obtained from three experiments.
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2

Purification of OMC32 Polypeptide from OMC Fraction

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The OMC fraction was extracted overnight at 4°C with 100 mM CAPS buffer (3-[cyclohexamino]-1-propanesulfonic acid; Sigma Chemical Co., St. Louis, MO), pH 10.5. The extracted solution was centrifuged for 30 min at 100,000 X g in a Beckman SW40 rotor which resulted in high-pH soluble and insoluble fractions. SDS-PAGE revealed that the 38–19kDa polypeptide family (termed as OMCrpf polypeptides) was solubilized by high-pH extraction whereas the 54, 50, and 45 kDa polypeptides remained associated with the high-pH insoluble particulate fraction [21 (link), 27 (link)]. The high-pH soluble, supernatant fraction was dialyzed and lyophilized. The purification of the 32kDa polypeptide (OMC32) from the high-pH soluble fraction was performed by continuous-elution SDS-PAGE on 12% acrylamide gels using a Model 491 Prep Cell (Bio-Rad Laboratories, Hercules, CA) following the method of Olson et al [26 (link)].
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3

Biochemical Reagents for DNA Analysis

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All metal chlorides, formamide, and CAPS buffer were purchased from Sigma-Aldrich. Tris base was purchased from J. T. Baker and glacial acetic acid was from Mallinckrodt Chemicals. Ethylenediaminetetraacetic acid (EDTA)-disodium salt was obtained from EMD Chemicals, Inc. The 2-Log DNA ladder, also called 1 kb Plus DNA ladder, 1 kb Extend DNA ladder, bacteriophage lambda DNA and M13mp18 DNAs were purchased from New England Biolabs. E. coli chromosomal DNA (D2001) was obtained from Sigma-Aldrich. Agarose was from Gold Biotechnology.
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4

Bk-343-HOPO and Bk-DTPA XAS Samples

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Bk-343-HOPO and Bk-DTPA XAS samples were previously prepared as part of samples containing both 249Bk and 249Cf (Deblonde, Kelley et al., 2018 ▸ ; Kelley et al., 2018 ▸ ), and details are included here for completeness. For both 343-HOPO and DTPA, samples containing both 249Bk(III) and 249Cf(III) (ratio Cf/Bk = 1.9) were prepared using a 249BkCl3 stock solution in 0.1 M HCl that had decayed for 510 days to allow for in-growth of the 249Cf daughter. The Bk/Cf samples were prepared using a metal:ligand ratio of 1:1.3 to ensure complete metal–ion complexation and assembled from aliquots of the metal stock solution with either a 343-HOPO stock solution in water or a DTPA stock solution at pH 4. The metal–ligand complexes were diluted with CAPS buffer (Sigma–Aldrich, BioUltra, >99%) to volumes of ∼65 µl to yield final concentrations of 0.11 mM for 249Bk and 0.20 mM for 249Cf. The pH values of the samples were buffered to 7–8 and each solution was loaded into separate, triply contained, aluminium holders (analogous to Pu) within ten days of synchrotron measurement.
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5

N-terminal Sequencing of Protein Complexes

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The N-termini of the heavy chain, light chain, and Protac-digested heavy chain were analyzed by Edman degradation [37 ]. Purified proteins were subjected to reducing 12% SDS-PAGE and electrotransfered overnight at 30 V in CAPS buffer (Sigma-Aldrich, Oakville, ON) onto Immobilon-PSQ PVDF membrane (Millipore, Billerica, MA). The membrane was incubated for 10 mins in 0.2% (w/v) Ponceau S (Sigma-Aldrich, Oakville, ON) in 1% (v/v) acetic acid and washed three times in Milli-Q water. It was then stored in a moist chamber and submitted for sequencing (The Advanced Protein Technology Centre, Hospital for Sick Children, Toronto, ON). Stained protein bands were excised and analyzed by the Procise Protein Sequencing System (Applied Biosystems, Foster City, CA) as previously described [38 ].
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6

Probing Cell Wall Pectins with Pectate Lyase and BAPTA

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Pectate lyase from Aspergillus (Megazyme, 180 U/mg, Cat # E-PCLYAN2) at 4.7U/mL (8uL of stock solution in 5mL of 50 mM CAPS buffer, Sigma-Aldrich A4926; pH 10)10 (link) and BAPTA calcium chelation at 2mM (Sigma Aldrich – Cat # A4926) treatments were performed on cell wall peels generated as described above. Treatments were carried out for 3 hours and 10 min, respectively, on the peels.
To screen for the effectivity of the treatments prior to vitrification, staining of non-treated, BAPTA- or PL- treated onion peels by a homogalacturonan-specific probe, Chitosan OligoSaccharide coupled with Alexa-488 (COS488) (Figure S6B) was performed based on a protocol provided by Jozef Mravec (personal communication): 1:1000 dilution from the mother solution kindly provided by Jozef Mravec (kept at -20C wrapped in foil) in 50 mM MES buffer pH 5.8 for 15 min. Peels were then washed with DI water 3 consecutive times before being mounted between a slide and coverslip and then screened by confocal laser scanning microscopy.53 (link)
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7

Biophysical Characterization of Protein Complexes

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All ITC studies were performed on a Microcal ITC 200 from GE Healthcare; the ITC 200 v1.26.1 software supplied by the manufacturer was used to record the data and Origin 7 v7.0552 was used for data analysis. CD spectra were recorded on a Jasco J-1500 CD Spectrometer Model 150 with EXOS liquid cooling system from Koolance Inc. with the supplied Spectra Manager software Version 2.12.00 using a Fisher Scientific Suprasil quartz microcell with 500 µL capacity, 45 mm height and an optical path length l of 10 mm at 10.00 ± 0.01 °C. UV-vis 43 were prepared as described; metal-free sodium hydroxide at 99.999% purity, and CHES, TAPS and CAPS buffer at ACS reagent grade quality were purchased from Sigma-Aldrich and used as received. All buffer solutions were prepared by standard methods at ambient temperature accounting for temperature differences at their intended use.
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