Precast sds polyacrylamide gel

Manufactured by Bio-Rad

Sourced in United States

Precast SDS-polyacrylamide gel is a laboratory equipment used for the separation and analysis of proteins based on their molecular weight. It is a pre-made, ready-to-use gel that simplifies the SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) process, reducing the time and effort required for gel preparation.

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Lab products found in correlation

Ip buffer, by Beyotime (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Bca kit, by Beyotime (3 mentions) Protein lysis buffer, by Beyotime (3 mentions) Protein a g beads 161 4023, by Bio-Rad (2 mentions) Anti rabbit secondary antibody, by Cell Signaling Technology (2 mentions) Cleaved caspase 3 antibody, by Cell Signaling Technology (2 mentions) Amersham hybond 0.45 pvdf membranes, by GE Healthcare (2 mentions) Anti p atr antibody, by Cell Signaling Technology (2 mentions) Anti atr antibody, by Cell Signaling Technology (2 mentions) P chk1 antibody, by Cell Signaling Technology (2 mentions) Hrp conjugated anti β actin antibody, by Proteintech (2 mentions) Clarity or clarity max western ecl blotting substrates, by Bio-Rad (2 mentions) Complete proteinase inhibitor, by Roche (1 mentions) Ab234, by Evrogen (1 mentions) Westar sun, by Cyanagen (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Chemidoc imager, by Bio-Rad (1 mentions) Sypro ruby protein gel stain, by Thermo Fisher Scientific (1 mentions) Nitrocellulose membrane, by Merck Group (1 mentions)

8 protocols using precast sds polyacrylamide gel

Transient Transfection and Western Blot Analysis of JellyOp Variants

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HEK293 cells were transiently transfected with JellyOp-mKate or JellyOp(K72T)-mKate as described above. Two days after transfection, cells were lysed in standard TNE buffer supplemented with cOmplete™ proteinase inhibitor (Roche). Lysate containing 25 ng protein was incubated in Laemmli buffer for 30 min at 37 °C and run on a 4–20% precast SDS-polyacrylamide gel (Bio-Rad). Protein was transferred to a polyvinylidene difluoride membrane (Immobilion). The membrane was blocked and stained in Tris-buffered saline supplemented with 0.1% Tween 20. The blocking solution (30 min) contained 5% non-fat milk. The primary antibody solution (overnight) contained mouse anti-GAPDH (1:4000, Fitzgerald, 10R-G109A) and rabbit anti-tRFP (1:1000, Evrogen, AB234). The secondary antibody solution (45 min) contained anti-mouse HRP (1:3000, Jackson Immuno Research, 115-035-146) and anti-rabbit HRP (1:3000, Jackson Immuno Research, 111-035-144). Membranes were washed for 10 min between and after incubation steps. Stained membranes were developed using Westar Sun (Cyanagen, XLS063.0250) and imaged on a ChemiDoc MP imaging system (Bio-Rad).

van Wyk M, & Kleinlogel S. (2023). A visual opsin from jellyfish enables precise temporal control of G protein signalling. Nature Communications, 14, 2450.

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Protein Characterization of Cell Lysate

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K562 cells were washed 3X with PBS (−/−) to remove serum protein and resuspended in serum-free RPMI-1640 at 2×10⁶ cells ml^-1. For the cell lysate control, cells were mixed with Halt™ Protease Inhibitor Cocktail, EDTA-Free (ThermoFisher), then underwent a 30-second liquid nitrogen snap freeze before thawing on ice for 10 minutes. The freeze-thaw process was repeated 5X. Cell lysate was centrifuged at 20,000xg for 15 min at 15°C and the supernatant was collected. Cells were processed with 7 and 9.5 μm gap microfluidic devices, plus a No device control. Device and No device samples, 2.5 ml each, were centrifuged at 200xg. 1.5 ml of supernatant was collected and centrifuged again. Only 1 ml of supernatant was collected to avoid contamination from cell debris. All samples were mixed with protease inhibitor and then concentrated 10-fold using a Vivaspin® 5 kDa molecular weight cut-off spin concentrator (Sigma-Aldrich). Supernatant proteins were characterized by protein gel electrophoresis using pre-cast SDS-polyacrylamide gel (Bio-Rad) according to manufacturer protocol. Sample loaded gel was run at constant voltage 200V. Gel was stained with SYPRO Ruby protein gel stain (Thermo Fisher) according to manufacturer protocol. Stained gel was imaged on a Bio-Rad ChemiDoc imager.

Liu A., Yu T., Young K., Stone N., Hanasoge S., Kirby T.J., Varadarajan V., Colonna N., Liu J., Raj A., Lammerding J., Alexeev A, & Sulchek T. (2019). Cell Mechanical and Physiological Behavior in the Regime of Rapid Mechanical Compressions that Lead to Cell Volume Change. Small (Weinheim an der Bergstrasse, Germany), 16(2), e1903857.

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Protein Stability Analysis in Organoids

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Organoids were harvested with ice-cold Cell Recovery Solution (Corning) containing EDTA-free protease inhibitor cocktail (Sigma-Aldrich) and PhosSTOP (Sigma-Aldrich), followed with 10-min incubation on ice. Following centrifugation, the supernatant was discarded and the pellet was washed once with Cell Recovery Solution. For protein stability and half-life analysis, organoids were treated with 10 μg/ml cycloheximide (Sigma-Aldrich) for the indicated time points The pellet was lysed with RIPA buffer supplemented with protease and phosphatase inhibitor cocktail on ice for 30 min, clarified at 15,000 RCF for 15 min at 4 °C, and stored at −20 °C. Protein lysates were separated on an 8–16% precast SDS polyacrylamide gel (Bio-Rad, CA, USA), transferred onto a nitrocellulose membrane (Millipore, MA, USA), and blocked in 5% (w/v) BSA in Tween 20-TBST. Blots were incubated with primary antibodies overnight at 4 °C, followed by incubation with IR-Dye secondary antibodies (Licor, AL, USA). Bands were detected and quantified with an Odyssey Imaging System (Licor).

Wan L., Lin K.T., Rahman M.A., Ishigami Y., Wang Z., Jensen M.A., Wilkinson J.E., Park Y., Tuveson D.A, & Krainer A.R. (2023). Splicing Factor SRSF1 Promotes Pancreatitis and KRASG12D-Mediated Pancreatic Cancer. Cancer discovery, 13(7), 1678-1695.

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Protein Expression Analysis of m6A Regulators

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The cells were extracted with protein lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail. Protein concentration was determined using the BCA Kit (Beyotime, China). Proteins (25–35 μg) were separated on a 10% polyacrylamide precast SDS gel (Bio-Rad) followed by blotting on PVDF membranes (Millipore Billerica, MA, USA). The membranes were probed with the following antibodies against: Zfp217 (Abcam, USA, #48133), METTL3 (Abcam, #195352), ALKBH5 (Abcam, #69325), PPARγ (Cell Signaling Technology, USA, #C26H12), aP2 (Cell Signaling Technology, #D25B3), FTO (Santa cruz, USA, #sc-271713), YTHDF2 (Proteintech, China, #24744-1-AP), β-actin (Abclonal, China, #AC026), LMNB1 (Abclonal, #A1910) and Tublin (Abclonal, #AC021). Secondary antibodies and detection were according to description previously. For IP experiments, cells were lysed in IP buffer (Beyotime, China) and incubated with antibodies followed by the pull-down with protein A/G beads for subsequent western blot analysis.

Song T., Yang Y., Wei H., Xie X., Lu J., Zeng Q., Peng J., Zhou Y., Jiang S, & Peng J. (2019). Zfp217 mediates m6A mRNA methylation to orchestrate transcriptional and post-transcriptional regulation to promote adipogenic differentiation. Nucleic Acids Research, 47(12), 6130-6144.

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Protein Extraction and Immunoprecipitation Assay

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The cells were extracted with protein lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail. Protein concentration was determined using the BCA Kit (Beyotime, China). Proteins (25–35 μg) were separated on a 10% polyacrylamide precast SDS gel (Bio-Rad, Richmond, CA, USA) followed by blotting on PVDF membranes (Millipore Billerica, MA, USA). For IP experiments, cells were lysed in IP buffer (Beyotime, China) and incubated with IP-grade antibodies followed by the pull-down with protein A/G beads (161-4023) (Bio-Rad, Richmond, CA, USA) for subsequent immunoblot analyses.
For pull-down assay, MODE-K cell lysates were collected and centrifuged at 8000 × g. The supernatant was transferred to another tube, and the cell debris was thoroughly discarded. Prewashed streptavidin beads were added into the supernatant, allowing 2 h preincubation with motion at 4°C to remove unspecific binding proteins. Purified mouse recombinant Nur77-LBD proteins were dissolved in lysis buffer. The lysates or recombinant proteins were incubated with compounds, followed by incubation with indicated doses of biotin-GPA for 6 h. Beads were washed with IP buffer for three times and boiled in SDS buffer.

Deng Z., Liu Q., Wang M., Wei H.K, & Peng J. (2020). GPA Peptide-Induced Nur77 Localization at Mitochondria Inhibits Inflammation and Oxidative Stress through Activating Autophagy in the Intestine. Oxidative Medicine and Cellular Longevity, 2020, 4964202.

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Protein Extraction and Immunoprecipitation Protocols

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The cells were extracted with protein lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail. The protein concentration was determined using the BCA Kit (Beyotime, China). Proteins (25–35 μg) were separated on a 10% polyacrylamide precast SDS gel (Bio-Rad, Richmond, CA, USA) followed by blotting on PVDF membranes (Millipore Billerica, MA, USA). For IP experiments, the cells were lysed in IP buffer (Beyotime, China) and incubated with IP-grade antibodies, followed by pull-down with protein A/G beads (161-4023) (Bio-Rad, Richmond, CA, USA) for subsequent immunoblot analyses.
For pull-down assay, THP-1 cell lysates were collected and centrifuged at 8000 × g. The supernatant was then transferred to another tube and the cell debris was thoroughly discarded. Prewashed streptavidin beads were added into the supernatant, allowing 2 h of preincubation with shaking at 4°C to remove unspecific binding proteins, followed by incubation with indicated doses of biotin-GPA for 6 h. Beads were washed with IP buffer for three times and boiled in SDS buffer.

Deng Z., Ni J., Wu X., Wei H, & Peng J. (2020). GPA peptide inhibits NLRP3 inflammasome activation to ameliorate colitis through AMPK pathway. Aging (Albany NY), 12(18), 18522-18544.

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Western Blot Analysis of Cell Signaling

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Tumor tissues were homogenized using a BeadBug Homogenizer (Benchmark Scientific, Sayreville, NJ). Protein lysates were prepared with lysis buffer (1% Triton X-100, 0.05% SDS, 100 mM Na₂HPO4, and 150 mM NaCl). Protein lysate was electrophoresed on a 4–20% pre-cast SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred onto Amersham Hybond 0.45 PVDF membranes (GE Healthcare, Chicago, IL). After blocking with 5% non-fat milk in PBS-0.05% Tween 20, the membranes were incubated with primary antibodies at 4°C overnight, and then secondary antibodies for 1 h at room temperature. Antibodies include anti-p-ATR antibody (#2853, Cell Signaling Technology), anti-ATR antibody (#13934, Cell Signaling Technology), p-CHK1 antibody (#2348, Cell Signaling Technology, Danvers, MA), cleaved-caspase-3 antibody (#9664, Cell Signaling Technology), HRP-conjugated anti-β-actin antibody (#HRP-6008, ProteinTech), anti-rabbit secondary antibody (#7074, Cell Signaling Technology). The blots were developed using Clarity or Clarity Max Western ECL Blotting Substrates (Bio-Rad).

Harold J., Bellone S., Manavella D.D., Mutlu L., McNamara B., Hartwich T.M., Zipponi M., Yang-Hartwich Y., Demirkiran C., Verzosa S.M., Choi J., Dong W., Buza N., Hui P., Altwerger G., Huang G.S., Andikyan V., Clark M., Ratner E., Azodi M., Schwartz P.E, & Santin A.D. (2022). Elimusertib (BAY1895344), a Novel ATR Inhibitor, Demonstrates in vivo Activity in ATRX Mutated Models of Uterine Leiomyosarcoma. Gynecologic oncology, 168, 157-165.

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Elimusertib Effects on DNA Damage Response

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Cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (Seradigm, Radnor, PA) and penicillin/streptomycin (Life Technologies) at 37°C and 5% CO₂. They were treated with a medium containing elimusertib (0.3 μM or 3 μM) or DMSO solvent (0.03%) which functioned as the untreated control. At the desired time points, cells were trypsinized and collected for preparing protein lysates. Protein lysates were prepared with lysis buffer (1% Triton X-100, 0.05% SDS, 100 mM Na₂HPO4, and 150 mM NaCl) and then electrophoresed on a 4-20% pre-cast SDS-polyacrylamide gel (Bio-Rad) and transferred onto Amersham Hybond 0.45 PVDF membranes (GE Healthcare, Chicago, IL). After blocking with 5% non-fat milk in PBS-0.05% Tween 20, the membranes were incubated with primary antibodies at 4°C overnight, and then secondary antibodies for 1 h at room temperature. Antibodies include anti-p-ATR antibody (#2853, Cell Signaling Technology), anti-ATR antibody (#13934, Cell Signaling Technology), p-CHK1 antibody (#2348, Cell Signaling Technology), cleaved-caspase-3 antibody (#9664, Cell Signaling Technology), HRP-conjugated anti-β-actin antibody (#HRP-6008, ProteinTech), anti-rabbit secondary antibody (#7074, Cell Signaling Technology). The blots were developed using Clarity or Clarity Max Western ECL Blotting Substrates (Bio-Rad).

Manavella D.D., McNamara B., Harold J., Bellone S., Philipp Hartwich T.M., Yang-Hartwich Y., Mutlu L., Zipponi M., Demirkiran C., Verzosa M.S., Altwerger G., Ratner E., Huang G.S., Clark M., Andikyan V., Azodi M., Schwartz P.E., Dottino P.R., Choi J., Alexandrov L.B., Buza N., Hui P, & Santin A.D. (2022). Ovarian and uterine carcinosarcomas are sensitive in vitro and in vivo to Elimusertib, a novel ataxia-telangiectasia and Rad3-related (ATR) kinase inhibitor. Gynecologic oncology, 169, 98-105.

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