Sds sample buffer

Manufactured by Bio-Rad

Sourced in United States, Italy

The SDS sample buffer is a solution used to prepare protein samples for electrophoresis. It denatures and solubilizes proteins, allowing for their separation based on molecular weight. The buffer contains sodium dodecyl sulfate (SDS), which coats the proteins with a uniform negative charge, and a reducing agent, which breaks down disulfide bonds.

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Lab products found in correlation

Nitrocellulose membrane, by Bio-Rad (8 mentions) β mercaptoethanol, by Merck Group (7 mentions) Pvdf membrane, by Bio-Rad (5 mentions) Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Protease inhibitor cocktail, by Roche (4 mentions) Blocking buffer, by LI COR (4 mentions) Chemidoc mp imaging system, by Bio-Rad (3 mentions) Anti β actin, by Merck Group (3 mentions) Fluorescent secondary antibody detection, by LI COR (3 mentions) Tris glycine gel, by Bio-Rad (3 mentions) Protease inhibitor cocktail, by Merck Group (3 mentions) 2 mercaptoethanol, by Merck Group (3 mentions) Phosphate buffered saline (pbs), by Merck Group (3 mentions) Hrp conjugated sheep anti human igg, by GE Healthcare (2 mentions) Protease inhibitor cocktail, by GenDEPOT (2 mentions) Odyssey infrared imager, by LI COR (2 mentions) Dynabeads m 280 sheep anti mouse igg, by Thermo Fisher Scientific (2 mentions) Myc monoclonal antibody, by Thermo Fisher Scientific (2 mentions) Chemidoc xr, by Bio-Rad (2 mentions) Clarity western ecl substrate, by Bio-Rad (2 mentions)

Spelling variants of this product

Sample buffer (188 citations) Laemmli SDS sample buffer (15 citations) SDS sample loading buffer (9 citations) 2x Laemmli SDS-PAGE sample buffer (7 citations) Tris-Glycine SDS Sample Buffer (4 citations) SDS-PAGE sample loading buffer (3 citations) Laemmli’s SDS sample buffer (3 citations) SDS-containing sample buffer (2 citations) Laemmli protein sample buffer for SDS-PAGE (2 citations) Reducing SDS-PAGE sample buffer (2 citations)

78 protocols using sds sample buffer

Co-Immunoprecipitation of Protein Complexes

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Co-IP was performed as following, briefly, cells were collected and lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDT, 1% Triton X-100, PMSF freshly added to a final concentration of 1mM, and 1x protease inhibitor cocktail). After quantification using a BCA protein assay kit (ThermoFisher, #23225), 1 mg of total protein were used for Co-IP and incubated for overnight with 2 µg of anti-MYC, anti-MAX antibodies, and normal rabbit IgG as a negative IP control, respectively. The mixtures were incubated for another 2–4 hr with protein A/G agarose beads, and then beads were washed at least 4 times, and treated and boiled for 10 min with 1x SDS sample buffer (Bio-Rad, #161–0737).
Cell lysates were also treated with equal volume of 2x SDS sample buffer and resolved by SDS-PAGE under denaturing conditions and transferred onto PVDF membranes (Bio-Rad, #162–0177). The membranes were blocked with 5% non-fat milk (LabScientific, #M0841) in 1x PBST at RT for 2 hr and incubated with primary antibody overnight at 4°C, followed by incubation with horse radish peroxidase-conjugated secondary antibodies for 1 hr at RT. Specific bands were visualized with enhanced chemiluminescence (ECL) substrate (ThermoFisher, #32106) and exposed onto films with an AX 700LE film processor (ALPHATEK).

Wang Z., Yang B., Zhang M., Guo W., Wu Z., Wang Y., Jia L., Li S., Xie W, & Yang D. (2018). LncRNA epigenetic landscape analysis identifies EPIC1 as an oncogenic lncRNA that interacts with MYC and promotes cell cycle progression in cancer. Cancer cell, 33(4), 706-720.e9.

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Immunoblotting with Anti-Human IgG

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Ghosts and immunoprecipitates were dissolved in 2× SDS sample buffer (Bio-Rad) and run on a 12% polyacrylamide gel under non-reducing conditions. The proteins on the gel were transferred onto a PVDF membrane, which was blocked with 5% milk in TBS with 0.1% Tween (TBST) for 1 h at room temperature. The membrane was incubated with 5 μg/mL MGD21 overnight at 4ºC, washed with TBST, and developed with HRP-conjugated sheep anti-human IgG (GE Healthcare, cat. no NA933) used in combination with a chemiluminescent substrate.

Tan J., Pieper K., Piccoli L., Abdi A., Perez M.F., Geiger R., Tully C.M., Jarrossay D., Maina Ndungu F., Wambua J., Bejon P., Fregni C.S., Fernandez-Rodriguez B., Barbieri S., Bianchi S., Marsh K., Thathy V., Corti D., Sallusto F., Bull P, & Lanzavecchia A. (2015). A LAIR-1 insertion generates broadly reactive antibodies against malaria variant antigens. Nature, 529(7584), 105-109.

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Co-immunoprecipitation Workflow for Protein Interactions

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Co-immunoprecipitations were conducted according to the protocol in [77 (link)]. Briefly, the protein concentration of E⁻Du145 and E⁻PC3 lysates were determined by Lowry assay (Biorad Dc assay cat: 500, Biorad, UK). Aliquots of cell lysates containing 500 μg protein were transferred to new tubes and the volume adjusted to 400 μL with lysis buffer. 30 μL packed Pierce Protein A/G Plus Agarose Beads (Life Technologies #20423) were combined with 500 μg whole cell lysate on a rocker at 4 °C for 1 h to clear the lysate. 500 μg cleared protein lysate and 5 μg/mL primary antibody were rocked overnight at 4 °C. Control samples had 5 μg isotype-matched control IgG added. 30 μL of fresh packed beads were added to the protein lysate with primary antibody and rocked at 4 °C for 1 h. Samples were centrifuged for 5 min at 10,000 × g. The beads were washed three times with 500 μL immunoprecipitation buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA, 1% Tween) before applying 25 μL 2× SDS sample buffer (Bio-Rad 161–0737) with 10% β-mercaptoethanol (Sigma-Aldrich M6250) and boiled at 100 °C for 5 min. The samples were centrifuged briefly at room temperature before loading with Prot/Elec tips (Bio-Rad 2239916) on a western blot.

Pollan S.G., Huang F., Sperger J.M., Lang J.M., Morrissey C., Cress A.E., Chu C.Y., Bhowmick N.A., You S., Freeman M.R., Spassov D.S., Moasser M.M., Carter W.G., Satapathy S.R., Shah K, & Knudsen B.S. (2018). Regulation of inside-out β1-integrin activation by CDCP1. Oncogene, 37(21), 2817-2836.

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Proteomic Analysis of α-Synuclein Interactome

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The BioID expression vector ligated with α-syn WT or A53T was transfected into SH-SY5Y cells. Biotinylation was induced by treatment with 50 μM biotin. Cells were washed with ice-cold PBS and harvested in the lysis buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2 mM EDTA, 1 mM DTT, 0.4% (w/v) SDS, and a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA)) 24 h after biotinylation. After sonication, the lysates were then centrifuged at 22,250× g for 20 min. Biotinylated proteins in the supernatants were captured on streptavidin beads by rotation overnight at 4 °C. The following day, the beads were pelleted and washed thrice with wash buffer A (50 mM HEPES, 500 mM NaCl, 1% (v/v) Triton X-100, 1 mM EDTA, and a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA)) and twice with wash buffer B (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA)). Beads were then mixed with 2× SDS sample buffer (Bio-Rad, Hercules, CA, USA) and boiled at 95 °C for 15 min. The reaction was verified via streptavidin immunoblot analysis; coomassie-stained gel was used, in combination with mass spectrometry, to identify the proteins.

Lee J.Y., Kim H., Jo A., Khang R., Park C.H., Park S.J., Kwag E, & Shin J.H. (2021). α-Synuclein A53T Binds to Transcriptional Adapter 2-Alpha and Blocks Histone H3 Acetylation. International Journal of Molecular Sciences, 22(10), 5392.

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Surface Biotinylation of Cells and Intestine

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Surface biotinylation of cell lines was performed as previously described (Silvis et al. 2009; Collaco et al. 2010) after treatment either with 8Br‐cGMP or linaclotide in the presence or absence of H7 (PKA inhibitor) or H8 (PKG inhibitor). Briefly, cells were washed twice with ice‐cold PBS containing 0.1 mmol/L CaCl₂ and 1.0 mmol/L MgCl₂ (PBS‐CM) and immediately placed on ice in a cold room. Rat intestine surface biotinylation was performed using a modified protocol as previously described (Ameen et al. 2003; Golin‐Bisello et al. 2005; Ameen and Apodaca 2007), immediately after treatment either with normal saline (pH7.4, 37°C) or linaclotide (5 μg/mL/loop) in vivo. Intestinal loops were incubated with freshly prepared Sulfo‐NHS‐SS‐biotin (1 mg/mL) in ice‐cold PBS‐CM (pH 8.0) in the cold room and processed as described (Golin‐Bisello et al. 2005). Cell surface and intestinal tissue biotinylated proteins were dissociated from streptavidin agarose by 2× SDS sample buffer (BioRad). Cell lysates (30 μg) and biotinylated samples were separated by SDS‐PAGE to detect CFTR by Western Blot analysis.

Ahsan M.K., Tchernychev B., Kessler M.M., Solinga R.M., Arthur D., Linde C.I., Silos‐Santiago I., Hannig G, & Ameen N.A. (2017). Linaclotide activates guanylate cyclase‐C/cGMP/protein kinase‐II‐dependent trafficking of CFTR in the intestine. Physiological Reports, 5(11), e13299.

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Histone Extraction and Purification Protocol

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Cells were harvested and washed twice with ice-cold PBS. PBS and subsequent buffers were supplemented with 5 mM sodium butyrate to maintain the histone acetylation levels. Cells were resuspended in Triton extraction buffer (PBS with 0.5% (v/v) Triton X-100, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.02% (w/v) NaN₃), at a density of 10⁷ cells/mL and lysed on ice for 10 min with gentle stirring. The lysates were then centrifuged at 6500× g for 10 min at 4 °C to spin down the nuclei. After removing the supernatants, pellets were resuspended in 0.2 N HCl at a density of 4 × 10⁷ nuclei/mL overnight at 4 °C. The following day, samples were rotated at 6500× g for 10 min at 4 °C to pellet the debris. Supernatants containing histone proteins were retained and neutralized with 2 M NaOH at 1/10 volume of the supernatant. After determining the protein content using the Bradford assay, the samples were mixed with 2× SDS sample buffer (Bio-Rad, Hercules, CA, USA) and boiled at 95 °C for 15 min.

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Subcellular Fractionation and Immunoblot Analysis of Pkd2-Deficient Cells

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Pkd^2+/− cells were transfected with PDE4C-Flag^{17 (link)} then incubated at room temperature for 30 min in protein extraction buffer containing 50 mM Tris-Cl, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 0.1% deoxycholic acid, 100 mM PMSF, and 1 tablet/10 ml protease inhibitor cocktail (Roche, Indianapolis, IN). Extracts were clarified by centrifugation at 12,000×g for 10 min, and the supernatant was fractionated by ultracentrifugation through a 5–40% sucrose gradient at 4°C for 3 h. Fourteen fractions were collected from the top of the gradient, and 15 µl of each fraction was mixed with 2×SDS sample buffer (Bio-Rad). SDS-PAGE analysis was performed with 4–15% Nu-PAGE gels. Proteins were transferred to nylon membranes, and immunoblot analysis was performed with antibodies against markers of Golgi (GM130, Sigma), endoplasmic reticulum (calnexin, Sigma), plasma membrane (CD44, Sigma), and primary cilia [acetylated α-tubulin, Sigma; intraflagellar protein 140 (IFT140), gift from Dr. William Snell, UT Southwestern]. Additional antibodies were directed against polycystin-2 (YCC2), AC5/6 (C17, Santa Cruz), AC5 (PAC-501AP, FabGennix), AKAP150 (Santa Cruz), PKA-RIIa (BD Transduction Laboratories), pCREB and phosphorylated PKA substrates (Cell Signaling).

Wang Q., Cobo-Stark P., Patel V., Somlo S., Han P.L, & Igarashi P. (2017). Adenylyl cyclase 5 deficiency reduces renal cyclic AMP and cyst growth in an orthologous mouse model of polycystic kidney disease. Kidney international, 93(2), 403-415.

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Western Blot Protocol for Cultured Neurons

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Cultured neurons were collected directly in × 2 SDS sample buffer (Bio-Rad). Samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes, probed with primary antibodies in 5% BSA/PBS plus 0.05% Tween 20 overnight at 4 °C or 1 h at room temperature, followed by Dylight fluorescent secondary antibodies for 1 h. Membranes were imaged using an Odyssey Infrared Imager (model 9120; LI-COR Biosciences). Protein bands were quantified using the ImageJ (NIH) “Gels” function, and all bands were normalized to their loading controls.

Zhu M., Cortese G.P, & Waites C.L. (2018). Parkinson’s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons. BMC Biology, 16, 100.

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Protein Extraction and Immunoblotting from Brain Tissues

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Human and mice brain tissues were harvested and homogenized in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, phosphatase inhibitor cocktail I and II (Sigma-Aldrich, St. Louis, MO, USA), and a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA) using a Diax 900 homogenizer. After homogenization, the samples were rotated at 4 °C for 30 min to ensure complete lysis. The lysates were mixed with 2× SDS sample buffer (Bio-Rad, Hercules, CA, USA) and then boiled at 95 °C for 15 min. Protein concentrations were quantified using a bicinchoninic acid kit (Pierce, Waltham, MA, USA) with BSA standards. Immunoblotting was performed with the antibodies of interest and visualized using chemiluminescence (Pierce, Waltham, MA, USA). Densitometric analysis of the immunoblot bands was performed using ImageJ software (NIH, http://rsb.info.nih.gov/ij/, downloaded on 2 March 2017). Information on the PD specimens used in this study has been described previously [43 (link)].

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Immunoblotting with Anti-Human IgG

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