A7030

Manufactured by Merck Group

Sourced in United States, Germany

The A7030 is a piece of laboratory equipment manufactured by Merck Group. It is designed to perform specific functions within a laboratory setting. The core function of the A7030 is to facilitate precise measurements and data collection. Further details about the intended use or capabilities of this product are not available.

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Lab products found in correlation

P9767, by Merck Group (6 mentions) Countess 2, by Thermo Fisher Scientific (6 mentions) R92tc 2, by Mesoscale (5 mentions) Bovine serum albumin (bsa), by Merck Group (5 mentions) R32ac 1, by Mesoscale (4 mentions) Lsm 780 confocal microscope, by Zeiss (4 mentions) Bca kit, by Thermo Fisher Scientific (3 mentions) H 1368, by Bachem (2 mentions) Nhs peg4 biotin, by Thermo Fisher Scientific (2 mentions) Nhs 800cw, by LI COR (2 mentions) P 6002, by Echelon Biosciences (2 mentions) Fn1 fibronectin, by Merck Group (2 mentions) D2650, by Merck Group (2 mentions) P5585, by Merck Group (2 mentions) P8920, by Thermo Fisher Scientific (2 mentions) Draq5, by Thermo Fisher Scientific (2 mentions) D9542, by Thermo Fisher Scientific (2 mentions) Prolong gold antifade mounting media, by Thermo Fisher Scientific (2 mentions) Trypan blue, by Thermo Fisher Scientific (2 mentions) Tryple solution, by Thermo Fisher Scientific (2 mentions)

87 protocols using a7030

Gradual Adaptation to Palmitic Acid

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Cells were seeded on 10 cm culture dishes so that the confluency at the starting day of adaptation was 80–90%. To start adaptation, all media was removed and replaced by growth media supplemented with palmitic acid (PA) (Sigma, P5585) and 1% fatty acid-free BSA as a carrier (Sigma, A7030). Adaptation was done using gradual increase in PA concentration MDA-MB-231 (50 µM, 200 µM and 400 µM, HCC1806 (200 µM and 400 µM), E0771 (200 µM, 400 µM, 500 µM) ensuring around 50% of cell death at each step. Parental cells were cultured in parallel using growth media supplemented only with 1% fatty acid-free BSA (Sigma, A7030).
For PA supplemented media, PA was first dissolved in absolute ethanol to obtain a 50 mM stock. To prepare the working concentrations, certain volumes of PA stock were added into 1%BSA growth media and incubated at 37 °C for 1 h. PA stock was stored at 4 °C and used for no longer than 2 weeks.

Liu X.Z., Rulina A., Choi M.H., Pedersen L., Lepland J., Takle S.T., Madeleine N., Peters S.D., Wogsland C.E., Grøndal S.M., Lorens J.B., Goodarzi H., Lønning P.E., Knappskog S., Molven A, & Halberg N. (2022). C/EBPB-dependent adaptation to palmitic acid promotes tumor formation in hormone receptor negative breast cancer. Nature Communications, 13, 69.

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Immunofluorescence Staining of LC3B

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Cells were seeded on 5 μg/ml FN (fibronectin; Sigma-Aldrich, F2006)-coated coverslips in 24-well plates at 40,000 cells per well in normal culture medium. After transfection or proper treatments, the cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, 15714) in PBS for 20 min, permeabilized with 0.2% Triton X-100 in PBS for 20 min, and blocked in 0.2% BSA (Sigma-Aldrich, A7030) in PBS for 20 min. For endogenous LC3B staining, cells were further fixed in methanol for 20 min at -20°C after the paraformaldehyde fixation. The cells were sequentially incubated with primary and secondary antibodies diluted with 0.2% BSA (Sigma-Aldrich, A7030) in PBS for 30 min at 37°C. Coverslips were mounted on glass slides using Fluoromount-G (Electron Microscopy Sciences, 17984-24). Cells were imaged using a Zeiss LSM780 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). The final composite images were created using ImageJ (NIH).

Jia R., Guardia C.M., Pu J., Chen Y, & Bonifacino J.S. (2017). BORC coordinates encounter and fusion of lysosomes with autophagosomes. Autophagy, 13(10), 1648-1663.

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Palmitoylation Assay in HEK293T Cells

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HEK293T cells were plated on poly-d-lysine–coated 24-well plates (Corning BioCoat; VWR) and transfected with 0.33 μg of substrate plasmid and 0.66 μg of pEF-BOS-HA (pEF) plasmid (either empty as a control or encoding the DHHC enzymes). Two μl of polyethylenimine (1 mg/ml stock) (linear polyethyleneimine molecular weight 25,000, catalog no.: 43896; Alpha Aesar) was added to the DNA mix, incubated for 20 min, and then added to the cells. Six h post-transfection, cells were washed once with PBS and incubated overnight with 100 μM of either palmitic acid (P0500; Merck) or C16:0-azide (75 (link)) in 300 μl of serum-free Dulbecco’s modified Eagle’s medium supplemented with 1 mg/ml defatted bovine serum albumin (A7030; Merck) at 37 °C.

Salaun C., Tomkinson N.C, & Chamberlain L.H. (2023). The endoplasmic reticulum–localized enzyme zDHHC6 mediates S-acylation of short transmembrane constructs from multiple type I and II membrane proteins. The Journal of Biological Chemistry, 299(10), 105201.

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Voltammetric Sensor Calibration for eCB Analysis

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The calibration of the voltammetric sensor against authentic eCBs was performed by using AEA (A0580, Merck KGaA, Darmstadt, Germany), 2-AG (A8973, Merck KGaA, Darmstadt, Germany) and AA (23401, Merck KGaA, Darmstadt, Germany) standards. To test the discriminating performance of the analytical system against eCB moieties, glycerol (G5516, Merck KGaA, Darmstadt, Germany) and ethanolamine (ETA; 411000, Merck KGaA, Darmstadt, Germany) were chosen. Bovine serum albumin (BSA, fatty acid free; A7030, Merck KGaA, Darmstadt, Germany), ultra-low melting agarose (A2576, Merck KGaA, Darmstadt, Germany), and Tris Acetate-EDTA buffer (TAE; T8280, Merck KGaA, Darmstadt, Germany) were used for probe functionalization.

Grasso S., Santonico M., Pennazza G., Zompanti A., Piccoli A., Bisogno T, & Maccarrone M. (2021). BIONOTE as an Innovative Biosensor for Measuring Endocannabinoid Levels. Sensors (Basel, Switzerland), 21(2), 489.

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Palmitate-Mediated Glucolipotoxicity in Islets and Min6 Cells

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palmitate (Sigma–Aldrich P9767) was dissolved in 50% ethanol at 65 °C and diluted in 10% fatty acid free BSA (Sigma–Aldrich A7030) to a concentration of 7 mM. The mixture was incubated at 37 °C for 1 h to allow for conjugation before diluting in culturing media to a final concentration of 0.5 mM. Control media was made by performing the same procedure with 50% ethanol and no palmitate.
For islets, control media (described above) contained 11 mM glucose with no added palmitate. Media for glucolipotoxic conditions contained 25 mM glucose with 500uM palmitate.
For Min6, control media (DMEM) contained 5.6 mM glucose with no added palmitate while glucolipotoxic conditions had 25 mM glucose and 500uM palmitate.

Good A.L., Cannon C.E., Haemmerle M.W., Yang J., Stanescu D.E., Doliba N.M., Birnbaum M.J, & Stoffers D.A. (2019). JUND regulates pancreatic β cell survival during metabolic stress. Molecular Metabolism, 25, 95-106.

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Immunostaining of Corneal Tissue

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Freshly excised corneas were washed in PBS and fixated in acetone (ZO-1) or 2% formaldehyde (8-OHdG) for 15 min on ice. To block nonspecific staining, corneas were incubated in 3% normal donkey serum (S30-100ML, Chemicon, Temecula, CA, USA), 1% bovine serum albumin (A-7030, Sigma-Aldrich, St. Louis, MO, USA), and 0.3% Triton X-100 (9002-93-1, Sigma-Aldrich) for permeabilization of cell membrane (8-OHDG) for 1 hour at room temperature (RT). Corneas were then stained with conjugated anti–ZO-1 antibody (1:100; Thermo Fisher Scientific) or anti–8-OHdG antibody (1:100; JACI, Nagoya, Japan) at 4°C overnight, followed by incubation with rhodamine-conjugated secondary antibody (8-OHdG) for 30 min at RT. Each step was followed by three thorough 5-min PBS washes. For nuclei staining, corneal whole mounts were incubated with 4′,6-diamidino-2-phenylindole (0.5 μg/ml; Dojindo Laboratories, Tokyo, Japan) for 5 min at RT. Corneal whole mounts were prepared using an aqueous mounting medium containing an anti-fading agent (Fluoromount/Plus, Diagnostic BioSystems, Pleasanton, CA, USA). Specimen slides were observed under a florescence microscope (Axioplan 2 imaging, Carl Zeiss Inc., Thornwood, NY, USA).

Yamaguchi T., Higa K., Yagi-Yaguchi Y., Ueda K., Noma H., Shibata S., Nagai T., Tomida D., Yasu-Mimura R., Ibrahim O., Matoba R., Tsubota K., Hamrah P., Yamada J., Kanekura K, & Shimazaki J. (2020). Pathological processes in aqueous humor due to iris atrophy predispose to early corneal graft failure in humans and mice. Science Advances, 6(20), eaaz5195.

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SARS-CoV-2 Spike RBD Binding Assay

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Recombinant biotinylated SARS-CoV-2 spike Spike-receptor-binding domain with a
C-terminal human IgG Fc domain fusion (referred to as Spike-RBD) was prepared as
previously described^{50 (link)}. Calu-3 cells
were grown in 96-well flat bottom plates until >50% confluent. Media was aspirated
and cells were washed once with PBS. Cells were then treated with TrypLE to release them
from the plate, RPMI 1640 media was added to dilute TrypLE, and cells were pelleted by
centrifugation at 200×g for five minutes. From this point on, all steps were
carried out on ice. Cells were incubated in 3% BSA (Sigma Aldrich A7030) in DPBS
(Sigma-Aldrich D8537) for 15 minutes to block and washed twice in 3% BSA in DPBS by
centrifugation at 200×g for five minutes in v-bottom plates, followed by
resuspension. Spike-RBD was diluted in 3% BSA to appropriate concentrations and incubated
with cells for 30 minutes on ice. Cells were then washed twice with 3% BSA in DPBS and
incubated with Anti-Strep PE-Cy7 (Thermofisher SA1012) at 5 μg/mL. Cells were
washed twice and subjected to flow cytometry on a FACS Celesta in HTS mode. Cells were
gated to exclude doublets and the median PE-Cy7 signal was calculated for each sample. The
gating strategy is shown in Supplemental
Figure 2. EC₅₀ values and their 95% confidence intervals were
calculated by fitting the RBD binding data into a Sigmoidal, 4PL model in Prism 6.

Samelson A.J., Tran Q.D., Robinot R., Carrau L., Rezelj V.V., Mac Kain A., Chen M., Ramadoss G.N., Guo X., Lim S.A., Lui I., Nunez J., Rockwood S.J., Wang J., Liu N., Carlson-Stevermer J., Oki J., Maures T., Holden K., Weissman J.S., Wells J.A., Conklin B.R., TenOever B.R., Chakrabarti L.A., Vignuzzi M., Tian R, & Kampmann M. (2021). BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2. bioRxiv.

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Quantifying 4-Hydroxynonenal Levels

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4-Hydroxynonenal (4-HNE) levels were measured using the ELISA method. Samples were homogenized and centrifuged to obtain total protein. 5 ug of protein was added to each well on a protein binding plate (28298-602; VWR, Ville Mont-Royal, Quebec, Canada) and incubated overnight at 4 °C. With washes between each step (5×), blocking buffer (5% BSA) was added for 1 h at room temperature (RT), followed by the primary antibody (393207, EMD Millipore) for 1 h at RT. Secondary antibody conjugated to horse radish peroxidase (7074; Cell Signaling Technology, Danvers, MA, USA) was added for 1 h at RT. TMB substrate solution was added for 20 min followed by stop solution (2M HCl). Absorbance was measured at 450 nm with 620 nm as a reference using a microplate reader (BioTek® Instruments). The absorbance was compared against that of a standard curve using known amounts of 4-HNE created by mixing 4-HNE (A8806; Sigma–Aldrich, Oakville, ON, Canada) with fatty acid free BSA (A7030; Sigma–Aldrich) as previously described (Weber et al., 2013 (link)).

Kim H.K., Isaacs-Trepanier C., Elmi N., Rapoport S.I, & Andreazza A.C. (2016). Mitochondrial dysfunction and lipid peroxidation in rat frontal cortex by chronic NMDA administration can be partially prevented by lithium treatment. Journal of psychiatric research, 76, 59-65.

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Flow Cytometry of dsRNA Detection

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Cells were trypsinized, washed with PBS and fixed with 4% formaldehyde diluted in PBS for 20 min at room temperature. Cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min followed by incubation in 1% BSA (Sigma, A7030) in PBS for 1 h. Primary antibodies (anti-dsRNA (J2) or normal mouse IgG2a (Iso)) were used at 2.5 μg ml⁻¹ in 1% BSA for 1 h at room temperature. Secondary donkey anti mouse IgG (H+L) conjugated with Alexa Fluor 488 were used at 2.2 μg ml⁻¹ concentration for 1 h at room temperature. Cells were rinsed three times with FACS buffer (0.5% BSA in PBS with 2mM EDTA). Data were acquired with a FACSCalibur (BD Biosciences) flow cytometer. Data were analysed in FlowJo (TreeStar).

Dhir A., Dhir S., Borowski L.S., Jimenez L., Teitell M., Rötig A., Crow Y.J., Rice G.I., Duffy D., Tamby C., Nojima T., Munnich A., Schiff M., de Almeida C.R., Rehwinkel J., Dziembowski A., Szczesny R.J, & Proudfoot N.J. (2018). Mitochondrial double-stranded RNA triggers antiviral signalling in humans. Nature, 560(7717), 238-242.

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Immunohistochemistry of Spinal Cord Neurons

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The deparaffinized, rehydrated sections of spinal cord and neurons fixed in 4% PFA were blocked with 5% bovine serum albumin (A7030, Sigma, Shanghai, China) for 30 min at room temperature and then incubated overnight at 4°C with primary antibodies against the following proteins: Brd4 (1:1000), Tuj1 (1:1000), Ace-tubulin (1:1000), Tyr-tubulin (1:1000), cleaved caspase-3 (1:1000), LC3 (1:1000), Lamp2 (1:1000), p-AMPK (1:500) and NeuN (1:1000). On the 2nd day, sections and fixed cells were incubated with Alexa Fluor FITC-, TRITC- or Cy5-conjugated donkey anti-rabbit/mouse/rat secondary antibodies (1:1000) for 60 min at 37°C, after which cellular nuclei were labeled with DAPI (36308ES20, Yeasen Biochemical, Shanghai, China). Images of different mice from the region of interest around the lesion margin (Figure 1B) and cells were blindly captured using a Nikon ECLIPSETi microscope (Nikon, Tokyo, Japan) with the same imaging setup.

Li Y., Xiang J., Zhang J., Lin J., Wu Y, & Wang X. (2020). Inhibition of Brd4 by JQ1 Promotes Functional Recovery From Spinal Cord Injury by Activating Autophagy. Frontiers in Cellular Neuroscience, 14, 555591.

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