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Du 800 spectrophotometer

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The DU-800 spectrophotometer is a laboratory instrument manufactured by Bio-Rad that measures the absorbance or transmittance of light by a sample over a specified wavelength range. It is designed to accurately quantify the concentration of various biomolecules, such as proteins, nucleic acids, and small molecules, in solution.

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Lab products found in correlation

Protein assay reagent, by Bio-Rad (26 mentions) Complete mini, by Roche (24 mentions) Protein a sepharose beads, by GE Healthcare (9 mentions) Protein a g sepharose beads, by GE Healthcare (4 mentions) A2220, by Merck Group (1 mentions) A2095, by Merck Group (1 mentions) A32953, by Thermo Fisher Scientific (1 mentions) B15002, by Selleck Chemicals (1 mentions) Protease inhibitor, by Roche (1 mentions) Prism 5, by GraphPad (1 mentions)

27 protocols using du 800 spectrophotometer

1

Protein Extraction and Immunoprecipitation

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Cell lysates were collected in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations of lysates were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, cells were treated with MG132 (10 μM) overnight after transfection 20 h, 1000 μg lysates were incubated with the indicated antibody (1–2 μg) for 3–4 h at 4°C followed by 1 h incubation with addition of carrier beads. Immunoprecipitants were washed five times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40), then were resolved by SDS-PAGE and immunoblotted with indicated antibodies.
Zhang L., Peng S., Dai X., Gan W., Nie X., Wei W., Hu G, & Guo J. (2017). Tumor suppressor SPOP ubiquitinates and degrades EglN2 to compromise growth of prostate cancer cells. Cancer letters, 390, 11-20.
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2

Protein Extraction and Immunoblotting

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Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations of lysates were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, 1000 μg lysates were incubated with the indicated antibody (1–2 μg) for 3–4 hours at 4 °C followed by 1 hour incubation with Protein A sepharose beads (GE Healthcare). Immunoprecipitants were washed five times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies. Quantification of the immunoblot band intensity was performed with ImageJ software.
Dai X., Gan W., Li X., Wang S., Zhang W., Huang L., Liu S., Zhong Q., Guo J., Zhang J., Chen T., Shimizu K., Beca F., Blattner M., Vasudevan D., Buckley D.L., Qi J., Buser L., Liu P., Inuzuka H., Beck A.H., Wang L., Wild P.J., Garraway L.A., Rubin M.A., Barbieri C.E., Wong K.K., Muthuswamy S.K., Huang J., Chen Y., Bradner J.E, & Wei W. (2017). Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4. Nature medicine, 23(9), 1063-1071.
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3

Cell Lysis and Immunoprecipitation Protocol

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Cells were lysed in CHAPS buffer (40 mM HEPES pH 7.4, 2.5mM MgCl2, 0.3% CHAPS) or EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The cell lysates were clarified by centrifugation at 13,000 r.p.m. at 4 °C for 10 min. The protein concentrations of lysates were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, 1000 μg lysates were incubated with 50% slurry of the affinity gel for 3 hours or the indicated antibody (1–2 μg) for 3–4 hours at 4 °C followed by one-hour incubation with Protein A sepharose beads (GE Healthcare). Immunoprecipitants were washed three times with CHAPS buffer containing 150 mM NaCl or NETN buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA and 0.5% NP-40) before being resolved by SDS-PAGE.
Gan W., Dai X., Dai X., Xie J., Yin S., Zhu J., Wang C., Liu Y., Guo J., Wang M., Liu J., Hu J., Quinton R.J., Ganem N.J., Liu P., Asara J.M., Pandolfi P.P., Yang Y., He Z., Gao G, & Wei W. (2020). LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control. Nature cell biology, 22(2), 246-256.
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4

Immunoprecipitation and Immunoblotting Protocol

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Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) (for immunoprecipitation) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The total protein concentrations of whole cell lysates were measured by a Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent. The same amounts of whole cell lysates were resolved by SDS–PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, 1000 μg lysates were incubated with the indicated antibody (1–2 μg) for 3–4 h at 4 °C followed by incubation for 1 h with Protein A Sepharose beads (GE Healthcare). Immunoprecipitants were washed five times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) before being resolved by SDS–PAGE and immunoblotted with indicated antibodies.
Liu P., Begley M., Michowski W., Inuzuka H., Ginzberg M., Gao D., Tsou P., Gan W., Papa A., Kim B.M., Wan L., Singh A., Zhai B., Yuan M., Wang Z., Gygi S.P., Lee T.H., Lu K.P., Toker A., Pandolfi P.P., Asara J.M., Kirschner M.W., Sicinski P., Cantley L, & Wei W. (2014). Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus. Nature, 508(7497), 541-545.
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5

Endogenous PTEN Methylation Assay

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Cells with indicated treatments were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations of whole cell lysates were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent. Equal amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitations analysis, 1000 μg lysates were incubated with the indicated antibody-conjugated beads for 4 h at 4 °C. The recovered immuno-complexes were washed four times with NETN buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA and 0.5% NP-40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies. For the endogenous PTEN methylation assays, EBC and NETN buffer containing 300 mM NaCl were used to disrupt the non-specific interacting proteins.
Zhang J., Lee Y.R., Dang F., Gan W., Menon A.V., Katon J.M., Hsu C.H., Asara J.M., Tibarewal P., Leslie N.R., Shi Y., Pandolfi P.P, & Wei W. (2019). PTEN Methylation by NSD2 Controls Cellular Sensitivity to DNA Damage. Cancer discovery, 9(9), 1306-1323.
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6

Cell Lysis and Immunoprecipitation Protocol

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Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (A32953, Thermo Fisher) and phosphatase Inhibitors (B15002, Bimake). The protein concentrations of lysates were measured using the Beckman Coulter DU-800 spectrophotometer and the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, cell lysates containing 1 mg of total proteins were incubated with anti-Flag agarose (A2220, Sigma) or anti-HA Agarose (A2095, Sigma) for 4 hours at 4 °C. Precipitants were washed three times with EBC buffer and resolved by SDS-PAGE followed by immunoblot analysis with indicated antibodies.
Chu X., Xiao X., Wang G., Uosef A., Lou X., Arnold P., Wang Y., Kong G., Wen M., Minze L.J, & Li X.C. (2023). Gasdermin D-mediated pyroptosis is regulated by AMPK-mediated phosphorylation in tumor cells. Cell Death & Disease, 14(7), 469.
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7

Immunoprecipitation and Immunoblotting Assay

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Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (Calbiochem 524624 and 524625). The protein concentrations of lysates were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, 1 mg lysates were incubated with the indicated antibody (1–2 μg) for 4 hr at 4 °C followed by 1 hr incubation with Protein A sepharose beads (GE Healthcare). Immunoprecipitates were washed five times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies.
Liu P., Gan W., Guo C., Xie A., Gao D., Guo J., Zhang J., Willis N., Su A., Asara J.M., Scully R, & Wei W. (2015). Akt-mediated Phosphorylation of XLF Impairs Non-homologous End Joining DNA Repair. Molecular cell, 57(4), 648-661.
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8

Lysis and Immunoprecipitation Procedure

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Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). Protein concentrations were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitations analysis, 1000 μg total cell lysates were incubated with the primary antibody-conjugated beads for 4 hours at 4 °C. The recovered immunocomplexes were washed four times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies.
Zhang J., Bu X., Wang H., Zhu Y., Geng Y., Nihira N.T., Tan Y., Ci Y., Wu F., Dai X., Guo J., Huang Y.H., Fan C., Ren S., Sun Y., Freeman G.J., Sicinski P, & Wei W. (2017). Cyclin D-CDK4 kinase destabilizes PD-L1 via Cul3SPOP to control cancer immune surveillance. Nature, 553(7686), 91-95.
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9

Immunoprecipitation and Western Blotting Protocol

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Cells were lysed in EBC buffer (50 mMTris pH 7.5, 120 mMNaCl, 0.5% NP-40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations of lysates were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, 1000 μg whole cell lysates were incubated with the indicated anti-Flag M2 affinity gel and monoclonal anti-HA agarose for 3–4 hr at 4 degrees Celcius (Millipore Sigma). Immunoprecipitants were washed five times with NETN buffer (20 mMTris, pH 8.0, 100 mMNaCl, 1 mM EDTA and 0.5% NP-40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies.
Saferali A., Yun J.H., Parker M.M., Sakornsakolpat P., Chase R.P., Lamb A., Hobbs B.D., Boezen M.H., Dai X., de Jong K., Beaty T.H., Wei W., Zhou X., Silverman E.K., Cho M.H., Castaldi P.J, & Hersh C.P. (2019). Analysis of genetically driven alternative splicing identifies FBXO38 as a novel COPD susceptibility gene. PLoS Genetics, 15(7), e1008229.
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10

Immunoprecipitation Assay Protocol

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Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations were measured by Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, CA). For immunoprecipitation assays, 1 mg whole cell lysate protein was incubated with the appropriate antibody-conjugated beads (8 ml) or antibody (1–2 mg) for 4 hours or overnight at 4°C. The recovered immuno-complexes were washed four times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies.
Zhang J., Chen M., Zhu Y., Dai X., Dang F., Ren J., Ren S., Shulga Y.V., Beca F., Gan W., Wu F., Lin Y.M., Zhou X., DeCaprio J.A., Beck A.H., Lu K.P., Huang J., Zhao C., Sun Y., Gao X., Pandolfi P.P, & Wei W. (2018). SPOP Promotes Nanog Destruction to Suppress Stem Cell Traits and Prostate Cancer Progression. Developmental cell, 48(3), 329-344.e5.
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