Protein lysis buffer

Manufactured by Bio-Rad

Sourced in United States

Protein lysis buffer is a solution used to extract and solubilize proteins from biological samples, such as cells or tissues. It is designed to disrupt cell membranes and denature proteins, allowing for the release and isolation of proteins for further analysis or purification.

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Lab products found in correlation

Bca kit, by Thermo Fisher Scientific (3 mentions) α tubulin, by Merck Group (2 mentions) Biomax mr film, by Kodak (2 mentions) Ecl plus reagent, by Merck Group (2 mentions) Anti phospho erk, by Merck Group (1 mentions) β actin, by Merck Group (1 mentions) Ab181602, by Abcam (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions) Ab182733, by Abcam (1 mentions) Ab182858, by Abcam (1 mentions) Ecl kit, by Cytiva (1 mentions) Tris buffered saline (tbs), by Bio-Rad (1 mentions) Ab13556, by Abcam (1 mentions) Anti gapdh antibody, by Cell Signaling Technology (1 mentions) Mmp 9, by Santa Cruz Biotechnology (1 mentions) Mmp 2, by Santa Cruz Biotechnology (1 mentions) Hrp conjugated goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions) Anti lamin b1, by Proteintech (1 mentions) Anti vinculin, by Proteintech (1 mentions) Hrp conjugated goat anti mouse igg, by Wuhan Servicebio Technology (1 mentions)

8 protocols using protein lysis buffer

Western Blot Analysis of ERK Activation

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MSCs at passage 3 and 6 were plated in 6-well plates at a density of 1 × 10⁵cells/cm² and starved in serum-free α-MEM medium for at least 6 h. Protein
lysis buffer (Bio-Rad, Hercules, CA, USA) was added, and thawed lysates were vortexed and
centrifuged. The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel
electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked
by incubation with 5% wt/vol nonfat dry milk. Membranes were then incubated with anti-ERK,
anti-phospho-ERK, and β-actin (Sigma) Abs at the appropriate dilutions overnight at 4°C.
After incubation, the membranes were washed in Tris-buffered saline containing Tween-20
(TBST). Secondary antibody conjugated to horseradish peroxidase was added to the membranes
in 5% nonfat dry milk in TBST. The negative control was used as described previously. The
western blotting assay was performed at least 3 times independently, representative
results are shown.

Zhang H., Li Z.L., Su X.Z., Ding L., Li J, & Zhu H. (2018). Subchondral bone derived mesenchymal stem cells display enhanced osteo-chondrogenic differentiation, self-renewal and proliferation potentials. Experimental Animals, 67(3), 349-359.

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Western Blot Analysis of Lung Proteins

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Lung tissues and cells were harvested and lysed by protein lysis buffer (Bio-Rad Laboratories). Then equal amount of protein samples (50 μg/lane) was separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by transferring onto polyvinylidene difluoride membranes. Blocked by 5% skimmed milk for 1 h at room temperature, the primary antibodies against TLR4 (ab13556; 1/500; Abcam, UK), Bax (ab182733; 1/2000; Abcam), Bcl-2 (ab 182,858; 1/2000; Abcam) and GAPDH (ab181602; 1/10000; Abcam) were incubated at 4 °C overnight. After washing by Tris-Buffered Saline (Bio-Rad Laboratories), the membranes were then cultured with horseradish peroxidase-conjugated secondary antibodies (1/10000; Abcam) at room temperature for 2 h. At last, the protein bands were assessed via an ECL kit (Amersham Biosciences) and the intensity was analyzed using ImageJ software. Original western blotting gels were provided in Additional file 1.

Sun Q., Luo M., Gao Z., Han X., Wu W, & Zhao H. (2021). Long non-coding RNA OIP5-AS1 aggravates acute lung injury by promoting inflammation and cell apoptosis via regulating the miR-26a-5p/TLR4 axis. BMC Pulmonary Medicine, 21, 236.

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Irradiation-Induced Stress Signaling in SSCs

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Normal mouse‐derived SSCs were seeded into cell culture plates (5 × 10⁵ cells/well) and irradiated with 2 Gy Co 60. FA was added to the SSC culture at the earliest possible time after irradiation. Nonirradiated SSCs were used as control. The SSCs were collected at 60 minutes postirradiation. Protein lysis buffer (BioRad, Hercules, California) was added, and the thawed lysates were vortexed and centrifuged. Proteins were separated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked by incubation with 5% wt/vol nonfat dry milk. The membranes were then incubated with anti‐phospho‐JNK (P‐JNK), anti‐JNK, anti‐phospho‐p38 (P‐p38), anti‐p38, anti‐phospho‐ERK (P‐ERK), anti‐ERK, and anti‐GAPDH antibodies (Cell Signaling Technology) at the appropriate dilutions overnight at 4°C. After incubation, the membranes were washed in Tris buffered saline with Tween‐20 (TBST). Horseradish peroxidase‐conjugated secondary antibodies were added to the membranes in 5% nonfat dry milk in TBST.

Liang J., Li P., Wang Q., Liao S., Hu W., Zhao Z., Li Z., Yin B., Mao N., Ding L, & Zhu H. (2021). Ferulic acid promotes bone defect repair after radiation by maintaining the stemness of skeletal stem cells. Stem Cells Translational Medicine, 10(8), 1217-1231.

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Western Blot Analysis of Protein Expression

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Cells were washed with PBS and lysed in protein lysis buffer (BioRad, Hercules, CA, USA) containing protease inhibitors. Cytoplasmic protein extracts were separated in 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes for 1 h at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies against LIN28A (1:500; NeoMarkers), MMP2 (1:500; Santa Cruz Biotechnology; mice) and (1:500; GeneTex Biotechnology; human), MMP9 (1:500; Santa Cruz Biotechnology; mice) and (1:500; GeneTex Biotechnology; human); and α-tubulin (1:2000; Sigma). After incubation with appropriate horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature, protein bands were detected using ECL-Plus reagent (EMD Millipore, Billerica, MA, USA) and Biomax MR Film (Kodak, Rochester, NY, USA), and relative protein expression was quantified by densitometry using the ImageQuant 5.2 software (Healthcare Bio-Sciences, Philadelphia, PA, USA).

Liu W.L., Chang J.M., Chong I.W., Hung Y.L., Chen Y.H., Huang W.T., Kuo H.F., Hsieh C.C, & Liu P.L. (2017). Curcumin Inhibits LIN-28A through the Activation of miRNA-98 in the Lung Cancer Cell Line A549. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(6), 929.

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Protein Extraction and Western Blot Analysis

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The total protein was extracted by protein lysis buffer (BioRad, Hercules, California) according to manufacturer's protocols. And then proteins were resolved via 10 % sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were incubated with anti-vinculin (proteintech, 1:5000), anti-phospho-YAP (CST, 1:2000), anti-YAP (CST, 1:2000), anti-COX2/Cyclooxygenase 2/Ptgs2 (proteintech, 1:1000), anti-Lamin b1 (proteintech, 1:2000) and anti-GAPDH (CST, 1:2000) at 4 °C for 8–12 h. After that, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Servicebio, 1:3000) or HRP-conjugated goat anti-mouse IgG (Servicebio, 1:3000) at room temperature for 1 h.

Li P.L., Chen D.F., Li X.T., Hao R.C., Zhao Z.D., Li Z.L., Yin B.F., Tang J., Luo Y.W., Wu C.T., Nie J.J, & Zhu H. (2023). Microgel-based carriers enhance skeletal stem cell reprogramming towards immunomodulatory phenotype in osteoarthritic therapy. Bioactive Materials, 34, 204-220.

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Western Blot Analysis of Cytoplasmic Protein Extracts

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Western blotting was performed as described previously [24 (link)]. Cells were washed with PBS and lysed in protein lysis buffer (Bio-Rad, Hercules, CA, USA) containing protease inhibitors. Cytoplasmic protein extracts were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 10% (v/v) polyacrylamide gels and transferred to polyvinylidene difluoride membranes for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies against HMGB1 (1:1,000; GeneTex), active β-catenin (1:500; Cell Signaling Technology), α-SMA (1:2,000; Abcam), vimentin (1:500, Santa Cruz Biotechnology), and α-tubulin (1:2,000, Sigma-Aldrich). After incubation with appropriate horseradish peroxidase-labelled secondary antibodies for 1 h at room temperature, protein bands were detected using ECL-Plus reagent (Millipore) and BioMax^® MR Film (Kodak, Rochester, NY, USA), and relative protein expression was quantified by densitometry using the ImageQuant^™ 5.2 software program (GE Healthcare Life Sciences, Philadelphia, PA, USA).

Liu P.L., Liu W.L., Chang J.M., Chen Y.H., Liu Y.P., Kuo H.F., Hsieh C.C., Ding Y.S., Chen W.W, & Chong I.W. (2017). MicroRNA-200c inhibits epithelial-mesenchymal transition, invasion, and migration of lung cancer by targeting HMGB1. PLoS ONE, 12(7), e0180844.

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Protein Extraction and Western Blot Analysis

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Total proteins were extracted using protein lysis buffer (BioRad, Hercules, CA) containing protease inhibitor cocktail (MCE). A protein extraction kit (Thermo) was used to extract nuclear and cytoplasmic proteins. The concentrations were determined using a BCA kit (Thermo). Equal amounts of protein from each sample were run on a 10% Tris–glycine SDS–PAGE gel, followed by transfer to PVDF membranes (Millipore). Membranes were blocked with 5% skim milk and probed with the indicated primary antibodies overnight at 4 °C. The membranes were washed 3 times with a mixture of Tris-buffered saline and Tween-20, followed by incubation with horseradish peroxidase-conjugated anti-IgG (Cell Signaling) for 1 h at room temperature. The intensities of the bands were visualized and determined using a Tanon-5200 automatic chemiluminescence imaging system (YPH-Bio, Beijing). The primary antibodies used were Akt, p-Akt, GAPDH (1:2000, Cell Signaling) and NRF2, KEAP1, GSK-3β, p-GSK-3β, lamin B1, GCLC, GCLM, HMOX1, and NQO1 (1:1000, ProteinTech). Quantification of the gray values of the bands was performed with ImageJ software.

Yin B.F., Li Z.L., Yan Z.Q., Guo Z., Liang J.W., Wang Q., Zhao Z.D., Li P.L., Hao R.C., Han M.Y., Li X.T., Mao N., Ding L., Chen D.F., Gao Y, & Zhu H. (2022). Psoralen alleviates radiation-induced bone injury by rescuing skeletal stem cell stemness through AKT-mediated upregulation of GSK-3β and NRF2. Stem Cell Research & Therapy, 13, 241.

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Western Blot Analysis of ERK5 and STAT3

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As described previously [14 (link),25 (link),26 (link)], the cultured cells were homogenized in protein lysis buffer (Bio-rad, Beijing, China), followed by protein concentration assessment with a BCA protein assay kit (R&D systems, Beijing, China). Western blot was performed with the following primary antibodies: rabbit anti-ERK5 (Cell signaling, San Jose, CA, USA), rabbit anti-pERK5 (Cell signaling), rabbit-anti-Stat3 (Cell Signaling) and rabbit anti-pStat3 (Cell Signaling). The secondary antibody was HRP-conjugated anti-rabbit (DAKO, Beijing, China). Image acquisition and densitometric analysis of the gels were performed with NIH ImageJ software (Bethesda, MA, USA).

Chen C., Wu S., Hong Z., Chen X., Shan X., Fischbach S, & Xiao X. (2019). Chronic hyperglycemia regulates microglia polarization through ERK5. Aging (Albany NY), 11(2), 697-706.

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