B9643

Manufactured by Merck Group

Sourced in Macao, Germany, Austria

B9643 is a laboratory centrifuge designed for general-purpose use in scientific research and clinical laboratories. The device is capable of separating and isolating various components of liquid samples through centrifugal force. Key technical specifications and features are available upon request.

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Lab products found in correlation

C2284, by Merck Group (3 mentions) Amyloglucosidase, by Merck Group (2 mentions) Ensight multimode plate reader, by PerkinElmer (2 mentions) Free glycerol reagent, by Merck Group (2 mentions) Tissuelyser lt, by Qiagen (2 mentions) Las 3000 instrument, by Fujifilm (1 mentions) β actin, by Fortis Life Sciences (1 mentions) Nucleospin rna protein kit, by Macherey-Nagel (1 mentions) Lc3a b, by Cell Signaling Technology (1 mentions) Glutamate assay kit, by Merck Group (1 mentions) Roswell park memorial institute (rpmi), by Dutscher (1 mentions) Hybond p, by GE Healthcare (1 mentions) Luminata crescendo western hrp substrate, by Merck Group (1 mentions) Goat anti mouse igg hrp sc 2005, by Santa Cruz Biotechnology (1 mentions) Expressplus page gel, by GenScript (1 mentions) Versadoc imaging system, by Bio-Rad (1 mentions) Ab108792, by Abcam (1 mentions) Aspergillus niger amylo α 1 4 α 1 6 glucosidase, by Roche (1 mentions) Glucose rtu kit, by bioMérieux (1 mentions) Tween 20, by Merck Group (1 mentions)

Spelling variants of this product

BCA1 and B9643 (7 citations)

12 protocols using b9643

Protein Isolation and Western Blot Analysis

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Proteins were isolated from cells and kidney tissue using NucleoSpin RNA/Protein kits (Macherey-Nagel). Protein concentration was calculated using the Bradford assay with bicinchoninic acid (B9643, Sigma) and copper sulfate (C2284, Sigma) solutions. Proteins were separated by 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (AE-6667-P, ATTO). The following primary antibodies were used in this study: Atg5 (#12294, Cell Signaling Technology), LC3 A/B (#12741, Cell Signaling Technology), p62 (#5114 and #23214, Cell Signaling Technology), Pten (#9552, Cell Signaling Technology), pDVL (ab124933, Abcam), Dvl2 (#3224, Cell Signaling Technology), and β-actin (A300-491A, Bethyl Laboratories).
Membranes were blocked with 5% skimmed milk in PBST (1 × PBS with 1% Tween-20), incubated with primary antibodies diluted in 1% skimmed milk with PBST overnight at 4°C, washed with PBST, and incubated with secondary antibodies in 2% skimmed milk for 1 h at room temperature. Protein bands were detected using enhanced chemiluminescent reagents (WSE-7120 EzWestLumi plus, ATTO) and band intensity was visualized using an LAS-3000 instrument (Fujifilm).

Mun H., Lee E.J., Park M., Oh G.T, & Park J.H. (2020). The Autophagy Regulator p62 Controls PTEN-Dependent Ciliogenesis. Frontiers in Cell and Developmental Biology, 8, 465.

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Protein Extraction from Pollen Grains

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100 mg pollen grains were mechanically decomposed by shaking pollen continuously at room temperature for three hours in PBS (phosphate buffered saline) [27 (link),28 ]. Afterwards the dispersion was centrifuged at 13.600 rpm for 5 min. Analysis of total soluble protein content was conducted with a classical BCA test [27 (link),28 ]. BCA solution and copper sulfate were obtained from Sigma (B9643; C2284). For the standard curve Albumin from Serva (11930) was used and its range was 25–1000 μg/ml concentration.

Jung S., Estrella N., Pfaffl M.W., Hartmann S., Handelshauser E, & Menzel A. (2018). Grass pollen production and group V allergen content of agriculturally relevant species and cultivars. PLoS ONE, 13(3), e0193958.

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Quantifying Brain Glutamate Levels

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The amount of brain glutamate (Glut) was quantified by Glutamate Assay Kit following the producer’s instructions (MAK004, Sigma-Aldrich, MO) [22 (link)], with some adaptation. Briefly, Cll was mechanically homogenate by a Dounce potter in Glutamate Assay buffer (200 uL each 10 mg tissue), the sample was centrifuged at 13,000 g for 15 min, and the supernatant collected for performing the test. Glutamate content in Hyper and Curc samples was expressed as fold change compared to Normo, after normalization for the total protein content in each sample, quantified by the Bicinchoninic Acid kit following the supplier’s instruction (B9643 and C2284, Sigma-Aldrich, MO). Data were expressed as mean ± S.D., and in fold vs. Normo (reference = 1).

Gazzin S., Dal Ben M., Montrone M., Jayanti S., Lorenzon A., Bramante A., Bottin C., Moretti R, & Tiribelli C. (2020). Curcumin Prevents Cerebellar Hypoplasia and Restores the Behavior in Hyperbilirubinemic Gunn Rat by a Pleiotropic Effect on the Molecular Effectors of Brain Damage. International Journal of Molecular Sciences, 22(1), 299.

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Generation of B16F10 Cell Extracts

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The murine melanoma cell line B16F10 was obtained from American Type Culture Collection (CRL-6475) and the luciferase and mCherry-expressing B16F10 cell line was kindly provided by Dr Mehdi Khaled (UMR 1299, Gustave Roussy Institute, Paris-Saclay University, France). Tumor cells were cultured in Roswell Park Memorial Institute medium (RPMI, Dutscher) supplemented with 10% of fetal calf serum (FCS, PAN-Biotech) and 1% HEPES (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), Dutscher) under an atmosphere containing 5% CO₂. An extract of B16F10 cells was obtained through three rounds of sonication at 60 W for 60 s. The resulting suspension was centrifuged at 2000 g for 15 min at 4°C and the protein concentration was determined using bicinchoninic acid (B-9643, Sigma) and cupric sulfate (C-2224, Sigma) and bovine serum albumin (BSA) as the protein standard. Extracts were then stored at −20°C.

Battistoni A., Lantier L., di Tommaso A., Ducournau C., Lajoie L., Samimi M., Coënon L., Rivière C., Epardaud M., Hertereau L., Poupée-Beaugé A., Rieu J., Mévélec M.N., Lee G.S., Moiré N., Germon S, & Dimier-Poisson I. (2023). Nasal administration of recombinant Neospora caninum secreting IL-15/IL-15Rα inhibits metastatic melanoma development in lung. Journal for Immunotherapy of Cancer, 11(5), e006683.

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Exosome Marker Expression Analysis

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Western blot analysis was performed to detect two positive markers (CD63 and HSP70) and to confirm the absence of two negative markers (GM130 and CytC). We first performed a BCA protein assay (B9643, C2284, Sigma) to quantify total protein in the samples. In each Western blot assay, after denaturation, 20 μg of total protein was loaded into an ExpressPlus PAGE Gel (GenScript). Proteins were transferred onto a PVDF membrane, Hybond-P (GE Healthcare Life Sciences). Incubation with primary antibodies (anti-CD63 rabbit IgG EXOAB-CD63A-1 [System Bioscience], anti-HSP70 mouse-monoclonal IgG sc32239 [Santa Cruz Biotechnology], anti-cytC mouse-monoclonal IgG sc-13156 [Santa Cruz Biotechnology] and anti-GM130 mouse-monoclonal IgG sc-55591 [Santa Cruz Biotechnology]) was carried overnight at 4 °C; all antibodies were diluted 1:1000 in 5% dry milk TBS-T. We diluted the secondary antibodies 1:5000 (goat anti-mouse IgG-HRP sc-2005 [Santa Cruz Biotechnology] and goat anti-rabbit IgG-HRP secondary antibody EXOAB-HRP [System Bioscience]), and they were incubated at RT for 1 hour. Finally, signals were detected with the chemiluminescence system Luminata Crescendo Western HRP Substrate (Millipore) and visualized using a Versadoc Imaging System (Bio-Rad).

Sanz-Rubio D., Martin-Burriel I., Gil A., Cubero P., Forner M., Khalyfa A, & Marin J.M. (2018). Stability of Circulating Exosomal miRNAs in Healthy Subjects. Scientific Reports, 8, 10306.

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Evaluation of Graft-Versus-Host Disease and Renal Function

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After transplantation, clinical GVHD scores were assessed weekly by a well-established standard scoring system incorporating weight loss, posture (hunching), mobility, fur texture, and skin integrity.^{19 (link)} Each parameter was graded between 0 and 2 followed by calculation of the cumulative score for each mouse. Blood samples were obtained immediately after euthanasia in week +4. Urine samples were taken the week before transplantation and in the third week after transplantation to measure protein (Pierce protein method with bicinchoninic acid solution (B-9643, Sigma-Aldrich, Munich, Germany) and copper(II) sulfate solution (C-2284, Sigma-Aldrich) and albumin (enzyme-linked immunosorbent assay (ELISA) #ab108792, Abcam, Cambridge, MA, USA) content as well as creatinine (enzymatic kinetic colorimetric method (creatinine peroxidase-antiperoxidase (PAP) test) #LT-CR-0101, Labor+Technik, Berlin, Germany) and N-acetyl-beta-D-glucosaminidase (NAG; ELISA #MBS703771, My BioSource, San Diego, CA, USA) levels. The albumin to creatinine ratio was calculated. Four weeks after transplantation, animals were euthanized, blood samples were taken, and creatinine as well as blood urea nitrogen (enzymatic ultraviolet method (urea UV test) #LT-UR-0010, Labor+Technik, Berlin, Germany) levels were measured to test renal function. After that, the kidneys were prepared for further investigation.

Schmid P.M., Bouazzaoui A., Schmid K., Birner C., Schach C., Maier L.S., Holler E, & Endemann D.H. (2017). Acute Renal Graft-Versus-Host Disease in a Murine Model of Allogeneic Bone Marrow Transplantation. Cell Transplantation, 26(8), 1428-1440.

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Glycogen Assay Protocol for Tissue Samples

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For GAA activity assays, the CNS, heart, liver, and skeletal muscles were rapidly dissected after euthanasia and PBS perfusion. Brains were sectioned into four coronal slabs of ~2 mm thickness and the spinal cord into two coronal slabs. All tissues were then snap-frozen in liquid nitrogen, and stored at −80 °C until biochemical analyses were performed. Tissues were homogenized in a phosphate buffer, homogenates were centrifuged at 13,000 rpm for 10 min at 4 °C and the resulting supernatant was assayed for GAA activity by measuring cleavage of 4-methylumbelliferyl-α-D-glucopyranoside aſter incubation for 1 h at 37 °C as previously described [46 (link)]. Protein concentration was measured using Bicinchoninic Acid method per manufacturer’s instructions (B9643, Sigma-Aldrich). Biochemical measurement of glycogen content was then performed as described elsewhere [20 (link)]. Tissue extracts were boiled for 3 min and incubated at 54 °C for 1 h in the presence or absence of Aspergillus niger amylo-α-1,4-α-1,6 glucosidase (5 U/ml; Roche, Mannheim, Germany) which converts glycogen to glucose. Samples were centrifuged and glucose level was determined in the supernatant using Glucose RTU kit (Biomerieux, Lyon, France) per manufacturer’s instructions.

Hordeaux J., Dubreil L., Robveille C., Deniaud J., Pascal Q., Dequéant B., Pailloux J., Lagalice L., Ledevin M., Babarit C., Costiou P., Jamme F., Fusellier M., Mallem Y., Ciron C., Huchet C., Caillaud C, & Colle M.A. (2017). Long-term neurologic and cardiac correction by intrathecal gene therapy in Pompe disease. Acta Neuropathologica Communications, 5, 66.

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Isolation and Culture of Mouse Embryonic Fibroblasts

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Mouse embryonic fibroblasts (MEFs) cells were isolated from day 13 to 15 embryos. MEF WT, SV40-immortalized MEFs (Simian vacuolating virus 40) and Gp130 ^F/F MEFs [53 (link)] were cultured in DMEM containing 15% FCS. The cells were typsinazed and washed with DMEM + 15% FCS before plating. Passage 3 cells with 1 ×10⁶ MEFs/well were seeded in 60 mm plates for 0-4 hours, 0.5 ×10⁶ MEFs/well for 24 hour and 0.25 ×10⁶ MEFs/well for 48 hour treatment with 5 ng/ml TGF-β respectively. After washing with cold PBS for two times, cells were lysed in ice-cold 200 μl RIPA lysis buffer, containing 1M Tris/HCL, 0.5 M EDTA, 5M NaCl, 10% Na Doc (Sodium Deoxycholate), 10% TX-100, 10% SDS, proteinase inhibitor 100 × and H ₂O. The cell lysates were passed through 27 G needle 5 times, then incubated on ice for 20 min. After incubation the samples were spun at 13,000 rpm for 30 min at 4 ^oC. The supernatant was transferred to new tubes: 20 μl of samples used for the BCA protein assay (Sigma kit B9643); 20 μl 5 × sample buffer was added to 80 μl of sample, the samples heated at 95 ^oC for 10 min and analysed by SDS-PAGE.

Khatibi S., Zhu H.J., Wagner J., Tan C.W., Manton J.H, & Burgess A.W. (2017). Mathematical model of TGF-βsignalling: feedback coupling is consistent with signal switching. BMC Systems Biology, 11, 48.

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Pollen Protein Quantification via BCA Assay

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Following Jung et al. [38 (link)], pollen grains were mechanically extracted. Total soluble protein content was quantified using BCA test [38 (link)]. The reagents (BCA solution and copper sulfate) were ordered from Sigma (B9643; C2284) and for the standard curve Albumin from Serva (11930) was used.

Jung S., Estrella N., Pfaffl M.W., Hartmann S., Ewald F, & Menzel A. (2021). Impact of elevated air temperature and drought on pollen characteristics of major agricultural grass species. PLoS ONE, 16(3), e0248759.

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Protein Quantification via Commercial BCA Assay

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Using a commercial BCA assay kit (Sigma, Austria, B9643) assay according to [40 (link)] the samples were incubated at 60 °C for 15 min to ensure the lowest protein-to-protein variations. After incubation, the samples were equilibrated for 10 min at room temperature prior to absorbance measurement within the linear range from 0.1 to 0.7 relative absorption units (rAU). The correlation between signal and protein concentration was established based on a separate calibration from 0.05 to 1 g/L BSA in NaOH/SDS. The limit of detection (LOD) was determined at 0.2 g/L.

Reichelt W.N., Waldschitz D., Herwig C, & Neutsch L. (2016). Bioprocess monitoring: minimizing sample matrix effects for total protein quantification with bicinchoninic acid assay. Journal of Industrial Microbiology & Biotechnology, 43, 1271-1280.

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