P8340 5ml

Manufactured by Merck Group

P8340-5ML is a laboratory product offered by Merck Group. It is a chemical solution with a volume of 5 milliliters. The core function of this product is to serve as a reagent or chemical standard for use in various laboratory applications. No further details about the intended use or specific applications of this product are provided.

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Lab products found in correlation

Ge10600023, by Cytiva (1 mentions) Mouse anti gfap, by Immunological Sciences (1 mentions) Clarity western ecl blotting substrate, by Bio-Rad (1 mentions) Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions) Alliance q9 advanced, by Uvitec (1 mentions) Sds page gel, by Thermo Fisher Scientific (1 mentions) Anti β actin 4970, by Cell Signaling Technology (1 mentions) Immobilon p, by Merck Group (1 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions) Mnase, by Thermo Fisher Scientific (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) R0278 50ml, by Merck Group (1 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Clarity western ecl substrate, by Bio-Rad (1 mentions)

6 protocols using p8340 5ml

Cerebellum Protein Expression Analysis

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Total lysates from both control and VPA-treated male mice cerebella (n = 6) were obtained using RIPA buffer (50 mM Tris pH 7.4; 1% NP-40; 0.5% Na deoxycholate; 0.1% SDS; 150 mM NaCl; 1 mM EDTA) supplemented with protease inhibitor cocktail (P8340-5Ml, Sigma Aldrich), 0.5 mM Na₃VO₄ and 1 mM DTT. Lysates were incubated on ice for 20 min, briefly sonicated and centrifuged for 10 min at 13,000 rpm, 4 °C. Protein extracts were quantified by Bradford assay, resuspended at a final concentration of 20 μg and analysed by electrophoresis through 8–10% SDS-PAGE and blotted on a PVDF membrane (GE10600023, Amersham). Blots were firstly incubated with a blocking solution (5% non-fat dry milk in PBS) for 1 h at 25 °C and then with the following primary antibodies: mouse anti-β-ACTIN (sc-47778, Santa Cruz Biotechnology), rabbit anti-ROR-α (NR1F1, Novus), mouse anti-SHANK2 (NBP2-12914, Novus), rabbit anti-SHANK3 (NBP2-41171, Novus), mouse anti-GFAP (MAB-94373, Immunological Sciences). Antibodies were diluted 1:1000 in PBS/5% BSA solution and incubated overnight at 4 °C. After washing, blots were incubated with HRP-linked anti-rabbit and anti-mouse secondary antibodies (GE Healthcare) diluted 1:10,000 in PBS/5% non-fat dry milk. ECL signal was developed using Clarity Western ECL Blotting Substrate (Biorad) and acquired with the Alliance Q9 Advanced (Uvitec Cambridge).

Guerra M., Medici V., Weatheritt R., Corvino V., Palacios D., Geloso M.C., Farini D, & Sette C. (2023). Fetal exposure to valproic acid dysregulates the expression of autism-linked genes in the developing cerebellum. Translational Psychiatry, 13, 114.

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Quantifying HDAC Protein Levels in Mouse Spleen

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Total proteins were extracted from spleen PMN- and M-MDSC sorted from spleens of EL4 tumor bearing mice. RIPA buffer supplemented with 1% of protease inhibitor cocktail (P8340-5ML; Sigma Aldrich) was used. Extracted proteins were resolved by electrophoresis using 10% SDS-PAGE gels (Invitrogen). 10 μg of protein was loaded per lane and proteins were electroblotted onto PVDF membrane (Immobilon-P, Milipore). The membranes were blocked with 5% non-fat dry milk in TBS plus 0.1% Tween20 (TBS-T) for 1 hr at room temperature, and then incubated with the primary antibodies in 5% non-fat dry milk in TBS-T overnight at 4°C. The primary antibodies used (dilution 1:1000) were anti-HDAC1 (5356S), anti-HDAC6 (7612S) and antiβactin (4970S) from Cell Signaling Technology, Inc., and anti-HDAC2 (32117), anti-HDAC3 (32369), anti-HDAC8 (187139) and anti-HDAC10 (108934) from Abcam. After washing, the membranes were incubated with HRP-linked secondary antibodies specific to rabbit and mouse antibodies (Cell Signaling Technology, Inc.) for 1 hr at room temperature. Detection was visualized by Pierce ECL Western Blotting Substrate (Thermo scientific).

Hashimoto A., Fukumoto T., Zhang R, & Gabrilovich D. (2020). Selective targeting of different populations of myeloid-derived suppressor cells by histone deacetylase inhibitors. Cancer immunology, immunotherapy : CII, 69(9), 1929-1936.

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Cell Lysis and Chromatin Preparation

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Cells (20-1000) were suspended in 1 μl PBS (14190136, Life Technologies Corporation) and mixed with 1 μl lysis buffer [4% Triton X-100, 100 mM Tris (pH 7.5), 100 mM NaCl, and 30 mM MgCl₂] containing freshly added 1% PIC (P8340-5ML, Sigma-Aldrich) and 1 mM PMSF (P7626-1G, Sigma-Aldrich). After 10-min incubation at room temperature, 0.2 μl of 500 mM CaCl₂ and 0.2 μl of 100U/μl MNase (88216, Life Technologies Corporation) were added followed by another 10 min incubation. The reaction was stopped by adding 0.222 μl of 0.5M EDTA and incubated on ice for 10 min. Chromatin solution was stored on ice before ChIP assay.

Deng C., Murphy T.W., Zhang Q., Naler L.B., Xu A, & Lu C. (2020). Multiplexed and ultralow-input ChIP-seq enabled by tagmentation-based indexing and facile microfluidics. Analytical chemistry, 92(20), 13661-13666.

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Western Blot Analysis of T1 and T2 Cells

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T1 and T2 cells were lysed and protein was isolated using Radioimmunoprecipitation assay (RIPA) lysis buffer (R0278-50ML, Sigma-Aldrich) with protease inhibitor (P8340-5ML, Sigma-Aldrich). The protein concentrations were quantified using BCA assay (23225, Thermo Fisher Scientific). 25 µg of total protein lysate was loaded on to 10% SDS gel and western blotting was performed as previously described (42 (link)). Antibodies used in the study are listed in Supplementary Table 5.

Thankamony A.P., Ramkomuth S., Ramesh S.T., Murali R., Chakraborty P., Karthikeyan N., Varghese B.A., Jaikumar V.S., Jolly M.K., Swarbrick A, & Nair R. (2023). Phenotypic heterogeneity drives differential disease outcome in a mouse model of triple negative breast cancer. Frontiers in Oncology, 13, 1230647.

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Western Blot Protein Detection Protocol

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U2OS cells were lysed in 10 mM KPO₄, 1 mM EDTA, 10 mM MgCl₂, 5 mM EGTA, 50 mM bis-glycerophosphate, 0.5% NP40, 0.1% Brij35, 0.1% sodium deoxycholate, 1 mM NaVO₄, 5 mM NaF, 2 mM DTT, and complete protease inhibitors (Sigma, P8340-5ML). Proteins were resolved by SDS-PAGE, transferred to PVDF (for low molecular weight proteins like LC3) or nitrocellulose membranes, blocked with 4% w/v non-fat dry milk in 1x TBS-T (TBS with 0.05% Tween 20), and probed with primary antibodies in blocking buffer at 4°C overnight followed by secondary antibodies in blocking buffer for 45 minutes at room temperature. Proteins were detected by chemiluminescence using ECL (100 nM Tris, pH 6.8, luminol (1.25 mM), p-coumaric acid (198 μM), H₂O₂ (0.009%)) or SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher, 34095).

Merrill N.M., Schipper J.L., Karnes J.B., Kauffman A.L., Martin K.R, & MacKeigan J.P. (2017). PI3K-C2α knockdown decreases autophagy and maturation of endocytic vesicles. PLoS ONE, 12(9), e0184909.

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Western Blot Protocol for Protein Analysis

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Cells were lysed with radio immunoprecipitation assay (RIPA) buffer (R0278-50ML, Sigma-Aldrich) containing 1X complete protease inhibitor cocktail (P8340-5ML, Sigma-Aldrich). Protein concentrations were determined using Pierce™ BCA Protein Assay Kit kit (23225, Thermofisher scientific, Waltham, Massachusetts, USA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto 45 μm polyvinylidene difluoride (PVDF) membrane (1620177, Biorad, Hercules, California, USA). Membranes were blocked using 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (P1379, Sigma) and probed with the respective primary and secondary antibodies. The signal was detected based on enhanced chemiluminescence (ECL) using Clarity Western ECL Substrate (1705061, Bio-Rad, Berkeley, CA, USA. β-actin was used as a protein loading control. Antibodies used in this study are listed in Table S5.

Thankamony A.P., Murali R., Karthikeyan N., Varghese B.A., Teo W.S., McFarland A., Roden D.L., Holliday H., Konrad C.V., Cazet A., Dodson E., Yang J., Baker L.A., George J.T., Levine H., Jolly M.K., Swarbrick A, & Nair R. (2020). Targeting the Id1-Kif11 Axis in Triple-Negative Breast Cancer Using Combination Therapy. Biomolecules, 10(9), 1295.

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