Cellular proteins were extracted with the Cell lysis buffer for IP (Beyotime). Proteins (500 μg) were incubated overnight at 4 °C with a rabbit anti-p27 antibody, or a control IgG. The immune complexes were immuno precipitated with Protein A/G Plus-Agarose beads (Santa Cruz), washed for three times with TBST, subjected to 12% SDS-PAGE, and detected with anti-CDK2, anti-Cylin A, or anti-p27 antibodies, respectively.
Cell lysis buffer for ip
Manufactured by Beyotime
Sourced in China
The Cell Lysis Buffer for IP is a solution designed to gently disrupt cell membranes and release cellular contents, including proteins, for subsequent immunoprecipitation (IP) experiments. The buffer contains a proprietary blend of detergents and other components that facilitate the extraction of target proteins while preserving their native structure and interactions.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g plus agarose, by Santa Cruz Biotechnology (3 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Sds page sample loading buffer, by Beyotime (1 mentions)
Mitochondria isolation kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Electrochemiluminescence western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Pierce silver stain for mass spectrometry kit, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Pierce classic magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
Ab155938, by Abcam (1 mentions)
Ab191527, by Abcam (1 mentions)
Hdac1, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
Hif 1α, by Proteintech (1 mentions)
Anti hif 1α, by Proteintech (1 mentions)
Vegfa, by Proteintech (1 mentions)
Anti hdac1, by Proteintech (1 mentions)
Control igg, by Cell Signaling Technology (1 mentions)
Fluorescein isothiocyanate isomer 1 fitc, by Merck Group (1 mentions)
Corn oil, by Merck Group (1 mentions)
10 protocols using cell lysis buffer for ip
1
Immunoprecipitation of Cell Cycle Proteins
2
Protein Extraction and Detection
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Total protein extracted by cell lysis buffer for IP (Beyotime, China) was incubated with antibodies and magnetic beads, binding proteins were extracted by 2×SDS-PAGE Sample Loading Buffer (Beyotime, China) and detected by WB.
3
Mitochondrial Protein Extraction and Analysis
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Mitochondria were extracted using a mitochondria isolation kit (Beyotime, China) according to the manufacturer’s instructions. Proteins were extracted using RIPA lysis buffer (Millipore, USA) supplemented with protease inhibitors and then centrifuged at 12,000 rpm (20 min, 4 °C). The protein samples were quantified via a BCA assay, separated via SDS‒PAGE, and transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes. Then, the membranes were blocked, incubated overnight with primary antibodies at 4 °C, and incubated for 1 h with conjugated secondary antibodies at room temperature. The protein bands were detected with electrochemiluminescence western blotting substrate (Thermo Fisher, USA), and the luminescence signals were measured with ImageJ software. The primary antibodies used are listed in Additional file 6 : Table S2.
To perform a coimmunoprecipitation (Co-IP) assay, the cells were lysed using Cell Lysis Buffer for IP (Beyotime) and incubated with Ig-A/G-magnetic beads (BioLinkedIn, China) preconjugated with antibodies against GPX4 (Santa Cruz, sc-166570) at 4 °C overnight. After extensive washing, the immunocomplexes were mixed with loading buffer and boiled for 10 min to elute the target proteins.
To perform a coimmunoprecipitation (Co-IP) assay, the cells were lysed using Cell Lysis Buffer for IP (Beyotime) and incubated with Ig-A/G-magnetic beads (BioLinkedIn, China) preconjugated with antibodies against GPX4 (Santa Cruz, sc-166570) at 4 °C overnight. After extensive washing, the immunocomplexes were mixed with loading buffer and boiled for 10 min to elute the target proteins.
4
Protein Interaction Analysis via IP-MS
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Immunoprecipitation (IP) and co-immunoprecipitation (Co-IP) were performed using Pierce Classic Magnetic IP/Co-IP Kit (Thermo Fisher Scientific, Grand Island, NY, USA) according to the manufacturer’s directions. Briefly, whole cell protein was extracted using cell lysis buffer for IP (Beyotime, Shanghai, China) supplemented with PMSF (Beyotime) and immunoprecipitated with GADD45g antibody (sc393261, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 µg per 200 µg of total protein). For Mass spectrometry assay, the elution products were separated by SDS-PAGE and stained with Pierce™ Silver Stain for Mass Spectrometry Kit (Thermo Fisher). The bands were cut from the silver-stained gel and analyzed via liquid chromatography tandem mass spectrometry (LC-MS/MS) in Shanghai Applied Protein Technology (Shanghai, China). For Western blot analysis, the antibodies used are as following: anti-GADD45g (sc393261, Santa Cruz, 1:500 dilution), anti-RAC2 (ab191527, Abcam, Cambridge, MA, USA, 1:1000 dilution) and anti-RAC1 (ab155938, Abcam, 1:1000 dilution).
5
β-catenin Immunoprecipitation from HUVEC Lysate
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HUVEC lysate was harvested using 200 μL Cell Lysis Buffer for IP (Beyotime biotechnology, Cat No. P0013) with protease and phosphatase inhibitors, and then immunoprecipitation was performed according to protocol provided by Millipore. In brief, 200 μL total proteins were firstly incubated with 2 μL β‐catenin antibody overnight at 4°C and subsequently mixed with 40 μL Pure ProteomeTM Protein A/G Mix Magnetic Beads at room temperature for 30 min. Using magnetic stand to capture the beads, then remove the supernatant sample and disengage the magnet and gently wash the beads with binding/wash buffer. After the last wash, disengage the magnet and add 1×loading buffer to denature the elution.
6
Validating TRPC3-SIRT1 Interaction by BLI
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GST pull‐down: The gene encoding TRPC3‐N (1‐369aa) and TRPC3‐C (659‐836aa) was synthesized by Detai Biologics Co., Ltd. HEK 293‐T cells were transfected with the above expression constructs to produce and purify recombinant protein for validating an interaction between TRPC3 and SIRT1 by bio‐layer interferometry (BLI).
Co‐immunoprecipitation (Co‐IP): Cells or cells transfected with indicated vectors were solubilized in cell lysis buffer for IP (Beyotime, China)17 with proteases and phosphatase inhibitors (pH 7.4). Pre‐cleared cell lysates were incubated with equal amounts of primary antibodies (2‐5 μL) or IgG at 4°C before performing the pull‐down with 50 μL of 1:1 Protein A/G PLUS‐Agarose (Santa Cruz Biotechnology, sc‐2003) for 2 hours. Beads were washed four times with lysis buffer, boiled and elution collected for WB analysis.18 , 19
Co‐immunoprecipitation (Co‐IP): Cells or cells transfected with indicated vectors were solubilized in cell lysis buffer for IP (Beyotime, China)
7
Western Blot and Co-IP Assay Protocol
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The western blot has been performed as described previously [17 (link)]. Primary antibodies against proteins were as follows: ATF3 (1:1000, Catalog number: ab207434, Abcam, UK), ZNF24 (1:1000, Catalog number: 11219-1-AP, Proteintech, Rosemont, USA), VEGFA (1:1000, Catalog number: 26157-1-AP, Proteintech, Rosemont, USA), HIF-1α (1:1000, Catalog number: 20960-1-AP, Proteintech, Rosemont, USA), HDAC1 (1:1000, Catalog number: 10197-1-AP, Proteintech, Rosemont, USA), and β-actin (1:1000, Catalog number: 20536-1-AP, Proteintech, Rosemont, USA).
The Co-IP assay has been performed as described previously [18 (link)]. Cells lysates were obtained by cell lysis buffer for IP (Beyotime). Take a small number of cell lysates for western blot analysis. IgG (Cell Signaling Technology) or primary antibodies: anti-HIF-1α (Proteintech, Rosemont, USA), anti-HDAC1 (Proteintech, Rosemont, USA) were added to the cell lysates, and incubated on a rotating platform at 4 °C overnight. Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) was added to the mixture and incubated with slow shaking at 4 °C for 2 h. Then separated the agarose, and analyzed the sample through western blot.
The Co-IP assay has been performed as described previously [18 (link)]. Cells lysates were obtained by cell lysis buffer for IP (Beyotime). Take a small number of cell lysates for western blot analysis. IgG (Cell Signaling Technology) or primary antibodies: anti-HIF-1α (Proteintech, Rosemont, USA), anti-HDAC1 (Proteintech, Rosemont, USA) were added to the cell lysates, and incubated on a rotating platform at 4 °C overnight. Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) was added to the mixture and incubated with slow shaking at 4 °C for 2 h. Then separated the agarose, and analyzed the sample through western blot.
8
Co-Immunoprecipitation of MZF1 and STUB1
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Cell lysates were prepared using Cell lysis buffer for IP (Beyotime) and adjusted to a final concentration of 1 μg/μL. 5% of cell lysates were retained as input group for direct Western blot analysis. For Co-IP group, protein A agarose beads were removed to the diluted cell supernatant and agitated for one hour at 4 °C to reduce the nonspecific background. Subsequently, cellular extracts were mixed overnight at 4 °C on a rotating platform with control IgG (Cell Signaling Technology) or following primary antibodies (4 μg/mL): rabbit anti-MZF1 (Santa Cruz Biotechnology), rabbit anti-STUB1 (Santa Cruz Biotechnology, Shanghai, China). Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) was then added and the mixture was incubated for a further 2 h at 4 °C with agitation. The agarose was then separated, rinsed with lysis buffer and equivalent volumes of each sample were assessed by Western blot analysis.
9
ATRA-Loaded Nanoparticle Cancer Therapy
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ATRA, acitretin, fluorescein isothiocyanate isomer I (FITC) and corn oil were acquired from Sigma-Aldrich (Missouri, USA). The 21-day ATRA sustained-release pellet was obtained from Innovative Research of America (Florida, USA). PLA–PEG–PLA triblock polymers (Mw 110 kDa, PEG 9%) were obtained from Jinan Daigang Biomaterial (Shandong, China). Cell lysis buffer for IP, BCA protein assay kit, protease inhibitor cocktail, 4′, 6-diamidino-2-phenylindole (DAPI) kit, hematoxylin and eosin (H&E) kit, crystal violet staining solution and citrate–EDTA antigen retrieval solution were procured from Beyotime Biotechnology (Shanghai, China).
Hepatocellular carcinoma cell lines were provided by the Cell Bank (Shanghai, China). Cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM; ThermoFisher, USA) containing 10% fetal bovine serum (FBS; PAN, Germany) and 1% penicillin–streptomycin solution (HyClone, USA) at 37°C under 5% CO2 in a humidified incubator. Male BALB/c nu/nu mice were obtained from Shanghai Laboratory Animal Center (SLAC, Shanghai, China). The animal care and experimental protocols were performed in accordance with and approved by the Experimental Animal Ethics Committee of Fujian Medical University.
Hepatocellular carcinoma cell lines were provided by the Cell Bank (Shanghai, China). Cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM; ThermoFisher, USA) containing 10% fetal bovine serum (FBS; PAN, Germany) and 1% penicillin–streptomycin solution (HyClone, USA) at 37°C under 5% CO2 in a humidified incubator. Male BALB/c nu/nu mice were obtained from Shanghai Laboratory Animal Center (SLAC, Shanghai, China). The animal care and experimental protocols were performed in accordance with and approved by the Experimental Animal Ethics Committee of Fujian Medical University.
10
Co-Immunoprecipitation Assay Protocol
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For Co-IP, original BC cells were extracted with cell lysis buffer for IP (Beyotime, Shanghai, China). 1000 μg protein of each sample was diluted in 500 μL IP lysis buffer. Specific antibodies (3 μg, ANXA9(Abcam, Cambridge, UK; Cat# ab166621), S100A4(Abcam, Cambridge, UK; Cat# ab197896) and HA-tag (Abclonal, Wuhan, China; Cat# AE008)) were incubated with the cell lysates and blended gently overnight at 4 °C. The homologous antibody with non-specific immunity (Rabbit pAb Control IgG, Abclonal, Wuhan, China, Cat# AC005; or Mouse pAb Control IgG, Abclonal, Wuhan, China, Cat# AC011) was used as control. Then 5 μL Protein A and 5 μL Protein G (absin, Shanghai, China) were added into each sample and blended gently for 4 h at 4 °C. After washing thrice in wash buffer (absin, Shanghai, China), captured proteins were eluted and denatured in 40 μL 1X SDS-PAGE Sample Loading Buffer (Beyotime, Shanghai, China) at 95 °C for 5 min. Finally, we used western blots to detect the binding proteins in these samples.
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