Protein loading buffer

Manufactured by Takara Bio

Sourced in Japan, United States, China

Protein loading buffer is a solution used to prepare protein samples for electrophoresis. It helps denature and solubilize proteins, allowing them to migrate through the gel during the electrophoresis process.

Automatically generated - may contain errors

Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Bcip nbt, by Merck Group (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Image lab software 5, by Bio-Rad (1 mentions) Chemidoc xps system, by Bio-Rad (1 mentions) Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions) Ripa lysis buffer, by Beyotime (1 mentions)

Spelling variants of this product

6× loading buffer (41 citations) 4× Protein SDS-PAGE Loading Buffer (5 citations) 2× protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer (3 citations)

4 protocols using protein loading buffer

Hybridoma Supernatant Western Blot

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The hybridoma culture supernatants were centrifuged at 1000× g for 5 min to remove cell debris, and then 20 μL supernatant was mixed with an equal volume of protein loading buffer (Takara, Japan). After denatured for 5 min in boiling water, the mixtures were subjected to SDS-PAGE (one was stained with Coomassie blue R-250, and the other was transferred to PVDF membranes (Millipore, Burlington, MA, USA)), and then blocked with 4% BSA at 4 °C overnight. After washed three times with PBST, the membranes were incubated with AP-conjugated goat-anti-mouse Ig at 37 °C for 1 h, and placed into the substrate solution containing nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate substrates (NBT/BCIP, Sigma, St. Louis, MO, USA) for staining.

Zhong Y., Tang X., Sheng X., Xing J, & Zhan W. (2018). Development and Characterization of Monoclonal Antibodies to the 32 kDa Viral Attachment Protein of Lymphocystis Disease Virus and Their Neutralizing Ability in Vitro. International Journal of Molecular Sciences, 19(9), 2536.

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Western Blot Analysis of Drosophila Proteins

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Drosophila embryos or larvae were homogenized in phosphate-buffered saline (PBS). Protein loading buffer (Takara, 9173) was added, and the mixture was heated for 5 min at 95°C. Protein samples were loaded onto a 15% SDS-PAGE gel for electrophoresis. After transfer to a PVDF membrane and blocking in 5% dried milk dissolved in tris-buffered saline containing 0.1% Triton X-100 (TBST), protein samples were incubated with primary antisera at 4°C overnight. After three washes with TBST for 10 min, membranes were incubated with secondary antisera from rabbit or mouse at room temperature for 1 h. After three washes with TBST for 10 min, and addition of enhanced chemiluminescence substrate, western blot signals were detected with a GE AI680UV instrument.

Zhang X., Wu X., Peng J., Sun A., Guo Y., Fu P, & Gao G. (2022). Cis- and trans-regulation by histone H4 basic patch R17/R19 in metazoan development. Open Biology, 12(11), 220066.

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Western Blot Analysis of Drosophila Proteins

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Drosophila larvae or adults were homogenized in buffer M (50 mM Tris-HCl pH 7.5, 50 mM KCl, 5% Glycerol, 1 mM DTT, 0.5 mM PMSF, and 0.5 mM EDTA). Nuclei were purified with Miracloth, transferred into 1.5 mL tubes, and centrifuged at 12000g for 15 min at 4°C. The supernatant was discarded and nuclei were resuspended with 100 μL buffer M and 10 μL 3 M KCl was added to increase the final KCl concentration to 100 mM. Following genomic DNA sonication, 30 μL of 4×protein loading buffer (Takara, Mountain View, CA, USA) was added and the mixture heated for 6 min at 95°C. Protein samples were loaded onto a 15% SDS-PAGE gel for electrophoresis. After transfer to PVDF membrane and blocking in 5% dried milk dissolved in TBST, protein samples were incubated with primary antisera at 4°C overnight. After three 10 min TBST washes, membranes were incubated with secondary antisera from rabbit or mouse at room temperature for 1 h. After three 10 min TBST washes, and addition of enhanced chemiluminescence substrate, western blot signals were detected with an ImageQuant LAS 4000 instrument.

Zhang W., Zhang X., Xue Z., Li Y., Ma Q., Ren X., Zhang J., Yang S., Yang L., Wu M., Ren M., Xi R., Wu Z., Liu J.L., Matunis E., Dai J, & Gao G. (2018). Probing the function of metazoan histones with a systematic library of H3 and H4 mutants. Developmental cell, 48(3), 406-419.e5.

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Immunoblot Analysis of TNFR2 Knockout Cells

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MC38/WT, MC38/TNFR2^-/-, CT26/WT, and CT26/TNFR2^-/- cell lysates were extracted by immunoprecipitation (RIPA) lysis buffer (Beyotime, Shanghai, China) containing 1 μM PMSF and 1% cocktail at 4 ^oC. The protein concentrations in each sample were determined using BCA protein assay kit (Thermo Scientific).
Each sample was denatured with 4 × protein loading buffer (Takara) at 100 °C for 10 min. Then, equal amounts of protein (20 μg per sample) were separated by 12% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF, Bio-Rad, USA) membranes. After blocking for 1 h in 5% skim milk powder in PBST at room temperature, the membranes were incubated with the primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. Protein bands were visualized by a hypersensitive chemiluminescence kit (Thermo Scientific) on ChemiDoc XPS system (Bio-Rad). Protein expressions were measured using the Bio-Rad Image Lab software 5.2.1.

Li P., Yang Y., Yang X., Wang Y., Chou C.K., Jiang M., Zheng J., Chen F, & Chen X. (2023). TNFR2 deficiency impairs the growth of mouse colon cancer. International Journal of Biological Sciences, 19(4), 1024-1035.

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