Cell lysis buffer

Manufactured by Bio-Rad

Sourced in United States

Cell lysis buffer is a solution used to disrupt the cell membrane and release the cellular contents, including proteins, nucleic acids, and other biomolecules. It is a fundamental tool in molecular biology and biochemistry experiments.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions) Bio plex 200 suspension array system, by Bio-Rad (3 mentions) Pro 2 wash station, by Bio-Rad (3 mentions) Bio plex 200 system, by Bio-Rad (3 mentions) Nitrocellulose membrane, by Merck Group (3 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions) Bio plex manager 6, by Bio-Rad (2 mentions) Bio plex 200 reader, by Bio-Rad (2 mentions) Bio plex pro cytokine 23 plex kits, by Bio-Rad (2 mentions) Precast gel, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Trypsin edta, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Phosphate buffered saline (pbs), by American Type Culture Collection (2 mentions) Non fat milk, by Bio-Rad (2 mentions) Anti bcl 2, by Merck Group (2 mentions) Anti bax, by Merck Group (2 mentions) Lipid peroxidation kit, by Abcam (2 mentions) β actin, by Merck Group (2 mentions)

Spelling variants of this product

Lysis buffer (41 citations) BioPlex cell lysis buffer (18 citations) Red cell lysis buffer (2 citations)

34 protocols using cell lysis buffer

Transcriptional Profiling and Protein Analysis

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cDNA was synthesised using High‐Capacity RNA‐to‐cDNA™ Kit (Applied Biosystems #4387406). qRT–PCR was performed using Taqman gene expression assay (Thermo Fisher #4304437) with following Taqman primers (Thermo Fisher): Hs01045540_g1 (ARC), Hs00156548_m1 (CLU), Hs00610256_g1 (DUSP1), Hs01044001_m1 (DUSP6), Hs00152928_m1 (EGR1), Hs00166165_m1 (EGR2), Hs00170630_m1 (FOS), Hs00171851_m1 (FOSB), Hs04187685_m1 (FOSL1), Hs00357891_s1 (JUNB), Hs00374226_m1 (NR4A1), Hs00943178_g1 (PGK1), Hs00169585_m1 (PPP1R15A), Hs00153133_m1 (PTGS2), Hs04334126_m1 (TFPI2), Hs00959047_g1 (TNFRSF12A), Hs00381614_m1 (ZCCHC12), Hs00185658_m1 (ZFP36).
Protein was extracted using Bio‐Rad Cell Lysis Buffer (#171‐304006M). Concentration was determined using Thermo Fisher Pierce BCA Protein Assay (#23228). 25–50 μg of purified protein was used for blotting. Images were acquired using Li‐Cor Odyssey Scanner. Western blot antibodies were as follows: EGR1 (Santa Cruz sc‐110), FOS (Cell Signaling #2250), CLU (Santa Cruz sc‐8354), FOSL1 (Santa Cruz sc‐376148).
For flow cytometry, cells were harvested 48 h after treatment and fixed in 2% paraformaldehyde (PFA) for 10 min at RT. Cells were permeabilised in methanol and incubated on ice for 30 min. For immunostaining, cells were incubated for 1 h with Cleaved Caspase‐3 rabbit mAb (Cell Signaling #9602).

Uhlitz F., Sieber A., Wyler E., Fritsche‐Guenther R., Meisig J., Landthaler M., Klinger B, & Blüthgen N. (2017). An immediate–late gene expression module decodes ERK signal duration. Molecular Systems Biology, 13(5), 928.

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Exosomal Protein Expression Analysis

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To compare the exosomal protein expression pattern after the azido sugar treatment, the isolated BC derived DMSO-exo, Man-exo, Gal-exo, and Glc-exo (10 μg) were lysed with cell lysis buffer (Cell Signaling, Danvers, MA), and electrophoretized in 4–12% gradient PAGE gel and stained by silver stain plus kit (Bio-Rad Inc, Hercules, CA).
For CD63 analysis, BC derived DMSO-exo, Man-exo, Gal-exo, and Glc-exo (20 μg) were lysed with cell lysis buffer, electrophoretized in 5–12% gradient PAGE gel. Standard Western blot procedures were performed. Primary antibody was rabbit anti-human CD63 antibody and secondary antibody was goat anti-rabbit HRP conjugated Ab (System Bioscience).
To check the integrin alpha 6 (ITGA6) expression, MCF7 and MDA-MB-231 cell lysates and MCF7 and MDA-MB-231 derived exosomes (20 μg) were electrophoretized in 8% PAGE gel and performed by standard western blot procedures. Primary antibodies were rabbit anti-human ITGA6 antibody and rabbit anti-human beat-actin antibody (Cell signaling). Secondary antibody was goat anti-rabbit HRP conjugated Ab (Santa Cruz Biotechnology).

Lee T.S., Kim Y., Zhang W., Song I.H, & Tung C.H. (2018). Facile Metabolic Glycan Labeling Strategy for Exosome Tracking. Biochimica et biophysica acta, 1862(5), 1091-1100.

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Brain Tissue Homogenization and Preservation

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Brains were homogenized (20% w/v) using a Mini Bead Beater (BioSpec Products) as previously described [6 (link),35 (link)] in ice-cold Cell Lysis Buffer (Bio-Rad) supplemented with 2 X Complete EDTA-free protease inhibitor cocktail (Roche) and 1 X cell lysis factors 1 and 2 (Bio-Rad) consisting of sodium orthovanadate and sodium fluoride to prevent dephosphorylation of proteins. Homogenates were stored at -80°C until use.

Carroll J.A., Race B., Williams K, & Chesebro B. (2018). Toll-like receptor 2 confers partial neuroprotection during prion disease. PLoS ONE, 13(12), e0208559.

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Protein Extraction and Western Blotting

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Protein extraction and Western blotting was performed similar to our previous work (37 (link)). Cells were washed with PBS, centrifuged, resuspended in cell lysis buffer (Bio-Rad, United States) and sonicated by a Bioruptor^® Pico sonication device (Diagenode, Belgium). From each sample either 20 or 30 μg of protein were loaded onto polyacrylamide gels for electrophoretic band separation and transferred to a nitrocellulose membrane (Bio-Rad, United States). Blocking of membranes was performed by incubation for 1 h in 5% blocking milk solution. The membranes were incubated together with the primary antibody overnight at 4°C on a rotator. After rinsing, the membranes were exposed to secondary IgG-HRP antibodies, ultimately being washed again and visualized with luminol-containing substrate solution or Clarity Western ECL Substrate (Bio-Rad, United States). Protein levels were normalized to GAPDH protein levels. Precision Plus Protein™ WesternC™ Blotting Standard (Bio-Rad) was utilized as a protein standard. Antibodies used for detection can be found in Table 3.

Schmidt A., Fuchs M., Stojanović S.D., Liang C., Schmidt K., Jung M., Xiao K., Weusthoff J., Just A., Pfanne A., Distler J.H., Dandekar T., Fiedler J., Thum T, & Kunz M. (2022). Deciphering Pro-angiogenic Transcription Factor Profiles in Hypoxic Human Endothelial Cells by Combined Bioinformatics and in vitro Modeling. Frontiers in Cardiovascular Medicine, 9, 877450.

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Protein Extraction and Western Blotting

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Protein extraction and Western blotting was performed similarly as in our previous work^{1 (link)}. Cells were washed with PBS, after which total protein was isolated from cell pellets using a cell lysis buffer (Biorad). Samples were pipetted onto polyacrylamide gels for electrophoretic band separation and transferred to a nitrocellulose membrane (Biorad). Blocking of membranes was performed by incubation for 1 h in 5% milk solution. The membranes were incubated with the primary antibody overnight. After rinsing, the membranes were exposed to IgG-HRP secondary antibodies, ultimately being washed again and visualized via the enhanced chemiluminescence (ECL) reagent (Biorad). Antibodies used for detection of QKI and GAPDH were as following: rabbit-anti-Qki (Sigma HPA019123) and mouse-anti-GAPDH (Abcam ab8245).

Stojanović S.D., Fuchs M., Liang C., Schmidt K., Xiao K., Just A., Pfanne A., Pich A., Warnecke G., Braubach P., Petzold C., Jonigk D., Distler J.H., Fiedler J., Thum T, & Kunz M. (2021). Reconstruction of the miR-506-Quaking axis in Idiopathic Pulmonary Fibrosis using integrative multi-source bioinformatics. Scientific Reports, 11, 12456.

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Tissue Protein Extraction and Quantification

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Samples were washed immediately with 1X cold phosphate-buffered saline (PBS) to remove blood and debris. Tissues were grossly dissected using a scalpel to remove scar and connective tissue, then stored at −80°C. Samples were ground cryogenically and lyophilized for 24 hours. For analysis, lyophilized tissue was thawed for 10 min at 4°C in 1 mL of cell lysis buffer (Bio-Rad, Hercules, CA) containing 20 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO). Protein extraction was performed using methods adapted from Hulse et al.^{40 (link)} Thawed samples were vortexed for 1–3 seconds and centrifuged at 5,000 × g for 5 minutes at 4°C. The supernatant was collected and tested for total protein content using a Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA), according to manufacturer’s instructions. Absorbance values for total protein content were determined on an Infinite M1000 multimode plate reader (Tecan, Raleigh, NC).

Prince N., Penatzer J.A., Shackleford T.L., Stewart E.K., Dietz M.J, & Boyd J.W. (2020). TISSUE-LEVEL CYTOKINES IN A RODENT MODEL OF CHRONIC IMPLANT-ASSOCIATED INFECTION. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 39(10), 2159-2168.

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Multiplex Cytokine Quantification in Rat Tissue

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To standardize samples for total protein content, tissue homogenates were individually diluted to a total protein concentration of 900 μg/mL with cell lysis buffer (Bio-Rad). Cytokine quantification was performed using a magnetic bead-based multiplex Rat Cytokine Th1/Th2 Kit (Bio-Rad) and measured using a Bio-Plex 200 suspension array system and Pro II Wash Station (Bio-Rad), according to the manufacturer’s instructions. The Th1/Th2 kit included the following cytokines: IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, GM-CSF, IFN-γ, and TNF-α. Results for these 11 cytokines were included in this analysis.

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Multiplex Immunomodulatory Protein Assay

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BAL cell pellets were re-suspended in cell lysis buffer (BioRad, Hercules, CA), sonicated for 20 seconds, and passed through a 1.2 μm filter before total protein concentration was determined by colorimetric assay (BioRad). The levels of 27 immunomodulating proteins were then assayed in BAL cell lysates using Bio-Rad Multiplex bead array following manufacturer guidelines as previously described (Bird et al., 2010 (link)). All samples were assayed in duplicate and the results were analyzed using the Bio-Plex manager software, version 5.0. All values were normalized to total protein concentration and reported as picograms of protein of interest per milligram of total protein.

O’Halloran E.B., Curtis B.J., Afshar M., Chen M.M., Kovacs E.J, & Burnham E.L. (2015). Alveolar macrophage inflammatory mediator expression is elevated in the setting of alcohol use disorders. Alcohol (Fayetteville, N.Y.), 50, 43-50.

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Quantifying CSF-1 in Tissue Lysates

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Tumors and mammary glands were prepared with BioRad cell lysis buffer and protein concentration of lysates was determined using the Pierce BCA Protein Assay Kit (Thermo Fischer Scientific) according to manufacturers’ recommendations. CSF-1 concentration in protein lysates was determined using Bio-Plex Pro™ Cytokine 23-Plex Kits (BioRad) and measured according to the manufactures’ instruction. Data acquisition and analysis was performed on a Bio-Plex 200 reader, using Bio-Plex Manager 6.0 software (Bio-Rad).

Salvagno C., Ciampricotti M., Tuit S., Hau C.S., van Weverwijk A., Coffelt S.B., Kersten K., Vrijland K., Kos K., Ulas T., Song J.Y., Ooi C.H., Rüttinger D., Cassier P.A., Jonkers J., Schultze J.L., Ries C.H, & de Visser K.E. (2019). Therapeutic targeting of macrophages enhances chemotherapy efficacy by unleashing type I interferon response. Nature cell biology, 21(4), 511-521.

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Quantifying CSF-1 in Tissue Lysates

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