Igepal ca 630

Manufactured by Thermo Fisher Scientific

Sourced in United States

IGEPAL CA-630 is a non-ionic surfactant. It is used as a wetting agent, emulsifier, and detergent in various industrial and laboratory applications.

Automatically generated - may contain errors

Lab products found in correlation

21 protocols using igepal ca 630

Reconstitution of Chromatin from Plasmids

Check if the same lab product or an alternative is used in the 5 most similar protocols

For low, medium, and high assembly degrees, 10 µg of plasmid library DNA (S. cerevisiae, S. pombe or E. coli) were mixed with ~2, 4, or 8 µg of Drosophila embryo histone octamers, respectively, in 100 µl assembly buffer (10 mM Tris·HCl pH 7.6, 2 M NaCl, 1 mM EDTA, 0.05% IGEPAL CA630 (Sigma), 0.2 µg/µl BSA). Samples were transferred to Slide-A-lyzer mini dialysis devices (MWCO 3.5 kDa; Thermo Fisher Scientific, cat no. 69550), which were placed in a 3 l beaker containing 300 ml of high salt buffer (10 mM Tris·HCl pH 7.6, 2 M NaCl, 1 mM EDTA, 0.05% IGEPAL CA630, 14.3 mM β-mercaptoethanol), and dialyzed against a total of 3 l low salt buffer (10 mM Tris·HCl pH 7.6, 50 mM NaCl, 1 mM EDTA, 0.05% IGEPAL CA630, 1.4 mM β-mercaptoethanol) added continuously while stirring via a peristaltic pump over a time course of 16 h. β-mercaptoethanol was always added freshly. After complete transfer of low salt buffer, samples were dialyzed against 1 l low salt buffer for 1 h at room temperature. DNA concentration of the SGD chromatin preparations was estimated with a DS-11+ spektrophotometer (Denovix). SGD chromatin could be stored at 4 °C for several weeks. To estimate the extent of the assembly degree, an aliquot of the sample was subjected to MNase digestion (below) for MNase-ladder read out.

Oberbeckmann E., Niebauer V., Watanabe S., Farnung L., Moldt M., Schmid A., Cramer P., Peterson C.L., Eustermann S., Hopfner K.P, & Korber P. (2021). Ruler elements in chromatin remodelers set nucleosome array spacing and phasing. Nature Communications, 12, 3232.

+ Open protocol

+ Expand

Cell-free binding studies of NRP-1 and TNC

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell-free binding studies were done as described in chapter 4.4 "Cell-free phage display", except 2 mg/mL of hexahistidine-tagged recombinant b1 domain of NRP-1 (inhouse) or C-domain of TNC (TNC-C; in-house) was used for coating. For NRP-1 b1, Tris buffer (50 mM, pH 7.0) containing 5 mM imidazole, 1 M NaCl and 0.05% Igepal CA-630 (all from Thermo Scientific Inc.), or for TNC-C and negative control NRP-1 b1 mutant (mut), PBS containing 0.05% Igepal CA-630 (Thermo Scientific Inc.), was used for the protein binding step. For incubations, 0.5 nM peptide-AgNPs were used, and eluted proteins with bound peptide-AgNPs were quantified by UV-vis spectrometry at 415 nm using a Nanodrop 2000c spectrophotometer (Thermo Scientific Inc.).

Tobi A., Haugas M., Rabi K., Sethi J., Põšnograjeva K., Paiste P., Jagomäe T., Pleiko K., Lingasamy P, & Teesalu T. (2024). Protease-activated CendR peptides targeting tenascin-C: mitigating off-target tissue accumulation. Drug delivery and translational research.

+ Open protocol

+ Expand

Dephosphorylation Assay in Cells and Hippocampi

Check if the same lab product or an alternative is used in the 5 most similar protocols

For dephosphorylation experiments, cells and hippocampi were homogenized in a buffer containing 100 mM NaCl and 50 mM Tris-Cl at pH 7.4 with 1% (v/v) IGEPAL^® CA-630 (ThermoFisher, Saint-Herblain, France) and a protease inhibitors cocktail without EDTA (Roche, Neuilly sur Seine, France) using either a “Precellys 24” (Bertin technologies, St Quentin-en-Yvelines, France) tissue homogenizer and followed by sonication with “vibracell 75 186” device (Sonics, Newton, CT, USA). Homogenates were centrifuged at 16,100 g for 20 min at 4°C with an Eppendorf 5415R centrifuge (Eppendorf, Hamburg, Germany), sonicated for 10 s and protein amounts normalized following a bicinchoninic acid protein assay (ThermoFisher). Samples were diluted to 1.0 mg/ml protein using homogenization buffer and incubated with 20 U/μl lambda phosphatase in MnCl₂ and enzyme buffer as supplied with the lambda protein phosphatase kit (New England Biolabs, Evry-Courcouronnes, France) for 3 h at 30°C. The reaction was stopped by the addition of sample buffer (Life Technologies) and heating to 95°C for 5 min. Control samples were treated identically without the addition of lambda phosphatase.

Chapelet G., Béguin N., Castellano B., Grit I., de Coppet P., Oullier T., Neunlist M., Blottière H., Rolli-Derkinderen M., Le Dréan G, & Derkinderen P. (2023). Tau expression and phosphorylation in enteroendocrine cells. Frontiers in Neuroscience, 17, 1166848.

+ Open protocol

+ Expand

ChIP-qPCR Assay for HIF-1α Binding

Check if the same lab product or an alternative is used in the 5 most similar protocols

Briefly, 5 × 10⁶ cells were fixed with 1% formaldehyde, quenched with 0.125 M glycine at room temperature, and then lysed in 500 μl lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, and 0.2% IGEPAL CA-630 (Thermo Fisher Scientific)) on ice for 30 min. The genomic DNA was sonicated into 200–500 bp. Ten percent of each whole-cell lysate was stored as input, and the rest of the lysate was incubated with 1 μg of the primary antibodies of HIF-1α (Cat. No. 36169, Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight. An additional 2-hour pull down was performed at 4°C with protein-A beads (Thermo Fisher Scientific). After washing by 600 mM LiCl, centrifuging at 800 g for 5 min and discarding the supernatant for three times, the purified DNA was used to perform qPCR to detect the enrichment of HIF-1α.

Pan J, & Zhao L. (2021). Long non-coding RNA histone deacetylase 4 antisense RNA 1 (HDAC4-AS1) inhibits HDAC4 expression in human ARPE-19 cells with hypoxic stress. Bioengineered, 12(1), 2228-2237.

+ Open protocol

+ Expand

EGFR Trafficking Assay Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

The EGFR trafficking assay was performed as previously described in Roberts et al. (2001) (link). In brief, siRNA-treated MDA–MB-231 cells were seeded into 10-cm dishes and grown to subconfluency on the day of the experiment. Cells were serum-starved 1 h before recycling in serum-free DMEM. Cells were biotinylated on ice in 10 mg sulfo-N-hydroxysuccinimide–SS-biotin per 75 ml PBS (21331; Thermo Fisher Scientific). Cells were then placed in serum-free DMEM 37°C for 30 min to internalize receptor–biotin complexes to equilibrium. Remaining cell surface biotin was stripped in 92-mM 2-mercaptoethanesulfonate sodium. Cells were placed again in serum-free DMEM for defined recycling periods at 37°C and subsequently stripped again and quenched with iodoacetamide. Cells were scraped and syringed in lysis buffer (200 mM NaCl, 75 mM Tris, 15 mM NaF, 7.5 mM EDTA, and EGTA, 1.5% Triton X-100, 0.075% Igepal CA-630, and Halt protease and phosphatase inhibitors [Thermo Fisher Scientific]). The levels of biotinylated receptor were measured using a sandwich ELISA with anti-EGFR antibody, streptavidin–horseradish peroxidase, and 0.56 mg/ml orthophenylenediamine. The percentage of recycled receptor was quantified as a percentage of the internal pool.

MacDonald E., Brown L., Selvais A., Liu H., Waring T., Newman D., Bithell J., Grimes D., Urbé S., Clague M.J, & Zech T. (2018). HRS–WASH axis governs actin-mediated endosomal recycling and cell invasion. The Journal of Cell Biology, 217(7), 2549-2564.

+ Open protocol

+ Expand

Immunofluorescence Analysis of DNA Damage Response

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Immunofluorescence experiments were performed on cells grown on 12-mm coverslips for 48 h after seeding. Cells were treated with 200 μg/ml phleomycin (Sigma-Aldrich) for 2 h, medium was replaced, and cells were further incubated at 37°C for the indicated times. Cells were washed with PBS or CSK buffer (10 mM 1,4-piperazinediethanesulfonic acid, pH 6.8, 100 mM NaCl, 3 mM MgCl₂, 300 mM glycerol) supplemented with 0.7% IGEPAL CA630 and 0.1 mg/ml RNase A (Thermo Fisher Scientific) for 3 min at room temperature (Britton et al., 2013 (link)). After two washes with ice-cold PBS, cells were cross-linked for 15 min with 4% paraformaldehyde at room temperature. Coverslips were blocked for 1 h at room temperature in blocking buffer (5% BSA in PBS). XRCC5 antibody was diluted 1:500 in blocking buffer and incubated for 2 h at room temperature. Anti-rabbit secondary antibody Alexa Fluor 488 (Thermo Fisher Scientific) was then added for 1 h at room temperature. Coverslips were counterstained with Hoechst 33342 (Thermo Fisher Scientific) and then mounted onto glass slides using ProLong Gold Antifade reagent (Thermo Fisher Scientific). Images were acquired using a Zeiss LSM 510 microscope.

Fantini D., Huang S., Asara J.M., Bagchi S, & Raychaudhuri P. (2017). Chromatin association of XRCC5/6 in the absence of DNA damage depends on the XPE gene product DDB2. Molecular Biology of the Cell, 28(1), 192-200.

+ Open protocol

+ Expand

Screening Cryptic Peptide Variants for uPA Cleavage

Check if the same lab product or an alternative is used in the 5 most similar protocols

To screen for uPA-cleavable cryptic PL3 peptide variants, we constructed a constrained phage library (configuration: AGRGRLVRXXXX, where X is a random amino acid). The phage library was cloned as described above, using partially randomized oligos (Table 1). Generated peptide-phage library pool was divided into two groups: non-treated and uPA pretreated. See below for uPA treatment protocol.
For cell-free phage display, 2 mg/mL of hexahistidinetagged recombinant b1 domain of NRP-1 (in-house) was coated onto Ni-NTA magnetic agarose beads (#36,113, Qiagen GmbH, Hilden, Germany) by incubating with endover-end mixing at RT for 1-1.5 h. Tris buffer (50 mM, pH 7.0) containing 5 mM imidazole, 1 M NaCl and 0.05% Igepal CA-630 (all from Thermo Scientific Inc.) was used for the dilutions; the same buffer with 0.1% bovine serum albumin (BSA; GE Healthcare, Little Chalfont, UK) was used for washes after the protein coating step. Proteincoated magnetic beads were incubated with phage library pools (5 × 10 9 pfu/mL) with end-over-end mixing for 1 h, followed by six washes with the washing buffer, and the release of protein-bound fraction with imidazole elution buffer (400 mM imidazole, 300 mM NaCl, 0.1% BSA and 0.05% Igepal CA-630 in PBS). Eluted phages were titered as described above.

+ Open protocol

+ Expand

Kinetics of IκBα Degradation

Check if the same lab product or an alternative is used in the 5 most similar protocols

DKO cells expressing ^3xFLAGIκBα were treated with 25 ng/ml TNFα for 15 min, and were collected and lysed in buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 1 mM DTT, 0.6% IGEPAL CA-630, protease inhibitors, and phosphatase inhibitors (Thermo Fisher Scientific). An aliquot of cell lysate (100 μl) was incubated with anti-FLAG beads for 30 min to get 0-hr sample. Another 100 μl aliquot was kept at room temperature for 9.5 hr and then incubated with anti-FLAG beads for 30 min to get the 10-hr control sample. For the rest of the lysate, recombinant Skp1ΔΔ•β-TrCP^139-569 (0.8x cell pellet volume of 12 μM Skp1•β-TrCP^139-569 solution, ~100x of endogenous β-TrCP level) was added as a chase, and the lysate was incubated at room temperature. Thirty min prior to each time point, 100 μl was drawn from the lysate-chase mixture and incubated with anti-FLAG beads for 30 min. All the proteins bound by the beads were eluted by 2x SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by Western Blot.

Liu X., Reitsma J.M., Mamrosh J.L., Zhang Y., Straube R, & Deshaies R.J. (2018). Cand1-mediated adaptive exchange mechanism enables variation in F-box protein expression. Molecular cell, 69(5), 773-786.e6.

+ Open protocol

+ Expand

Chromatin Immunoprecipitation Assay for NR5A2

Check if the same lab product or an alternative is used in the 5 most similar protocols

Briefly, 5 × 10⁶ cells were fixed with 1% formaldehyde, quenched with 0.125 M glycine at RT, and then lysed in 500 μL of lysis buffer (10 mM Tris-HCl [pH] 8.0, 10 mM NaCl, and 0.2% IGEPAL CA-630 [Thermo Fisher Scientific]) on ice for 30 min. Genomic DNA was sonicated to 200–500 bp. Ten percent of each whole-cell lysate was stored as the input, and the rest of the lysate was incubated with 1 μg of primary antibodies against NR5A2 (ab18293, Abcam, Cambridge, MA, USA), DNMT1, H3K27ac (NBP2-54615, Novus), H3K27me3 (NBP2-16840, Novus), mono-methylation of histone H3 at lysine 4 (H3K4me1) (NB21-1021, Novus), and ESR1 (ab32063, Abcam) at 4°C overnight. Then, an additional 2-h pull-down was performed at 4°C using protein-A beads (Thermo Fisher Scientific). Primers designed to encompass approximately 150 bp around the target GnRH genomic regions were used to detect the enrichment of histone deacetylases (HDACs) using qPCR.

Shen Y., Zhao H., Zhang L., Hu Y., Cai L., Li J, & Zhou S. (2021). The roles of DNA methylation and hydroxymethylation at short interspersed nuclear elements in the hypothalamic arcuate nucleus during puberty. Molecular Therapy. Nucleic Acids, 26, 242-252.

+ Open protocol

+ Expand

HSV-1 Infection Inhibition in HepaRG Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

HepaRG cells (1 × 10⁴) were seeded in a 96-well plate in William’s E medium and incubated overnight. Cells were infected with HSV-1 recombinant KOS6β at an MOI of 5, and the aurora kinase inhibitors (10 μM) were added at respective time points during infection: pretreatment (30 min prior to infection), 3 hpi, and 6 hpi. At 24 hpi, samples were harvested in 1% IGEPAL lysis buffer (IGEPAL CA-630; Thermo Fisher Scientific), and cell lysates were processed in a 1× CPRG solution (10X CPRG solution: 80 mM chlorophenol red-β-d-galactopyranoside, 0.6 M NaHPO₄, 0.4 M NaH₂PO₄, 0.1 M KCl, 0.01 M MgSO₄, 0.5 M 2-mercaptoethanol). Absorbance was measured at 595 nm 1 h post-addition of CPRG solution using a spectrophotometer (BioTEK Synergy, BioTek Instruments). The means and standard deviations for samples were determined from three independent experiments.

Ly C.Y., Pfannenstiel J., Pant A., Yang Z., Fehr A.R., Rodzkin M.S, & Davido D.J. (2022). Inhibitors of One or More Cellular Aurora Kinases Impair the Replication of Herpes Simplex Virus 1 and Other DNA and RNA Viruses with Diverse Genomes and Life Cycles. Microbiology Spectrum, 11(1), e01943-22.

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!