Cells were lysed in IP Lysis/Wash Buffer, with Proteinase Inhibitor Cocktail (MedChemExpress) added before use. The resulting lysate was then pre–cleaned using 25‐µL protein G sepharose beads at 4°C. Immunoprecipitation mixtures, including protein lysates, blocked protein G sepharose and the indicated antibodies or IgG control derived from the same species as the indicated antibody were incubated on a rotating wheel at 4°C overnight. Sepharose beads with the bound immunoprecipitates were collected and subjected to four washes with the cold IP Lysis/Wash Buffer and then analyzed by western blot assay.
Proteinase inhibitor cocktail
Manufactured by MedChemExpress
Sourced in United States, China
Proteinase Inhibitor Cocktail is a laboratory reagent designed to inhibit the activity of a wide range of proteolytic enzymes. It is formulated to provide broad-spectrum protection against protease-mediated degradation of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Protein g sepharose bead, by GE Healthcare (2 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence kit, by Wanlei (1 mentions)
C myc, by Abmart (1 mentions)
Hif 1a, by Santa Cruz Biotechnology (1 mentions)
P jak2, by Abcam (1 mentions)
β actin, by Affinity Biosciences (1 mentions)
Gapdh, by Affinity Biosciences (1 mentions)
Cyclin d1, by Proteintech (1 mentions)
P stat3, by Abcam (1 mentions)
Protein a g sepharose beads, by GE Healthcare (1 mentions)
Polyvinyl difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescent autoradiography, by Merck Group (1 mentions)
Hspa8, by Cell Signaling Technology (1 mentions)
Ai600 system, by GE Healthcare (1 mentions)
Gapdh 60004 1 ig, by Proteintech (1 mentions)
Ripa lysis buffer, by Wuhan Servicebio Technology (1 mentions)
Bca kit, by Wuhan Servicebio Technology (1 mentions)
Cyclin d1 antibody, by Proteintech (1 mentions)
10 protocols using proteinase inhibitor cocktail
1
Immunoprecipitation and Western Blot Analysis
2
Western Blot Analysis of JAK-STAT Pathway
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Differentiated cells were harvested and lysed in RIPA buffer supplemented with proteinase inhibitor cocktail (MedChemExpress). Proteins were separated on 10% SDS-PAGE gel and electro-transferred to PVDF membranes (Millipore). The membranes were blocked in blocking solution (5% skim milk in PBS) for 1 h at room temperature, and incubated overnight at 4°C with primary antibodies against JAK2 (1:1000, Cell Signaling Technology), p-JAK2 (Tyr1007/1008, 1:1000, Abcam), STAT3 (1:2000, Cell Signaling Technology), p-STAT3 (Tyr705, 1:2000, Abcam), HIF-1a (1:500, Santa Cruz Biotechnology), cyclin D1 (1:5000, proteintech), c-Myc (1:1000, Abmart), GAPDH (1:2000, Affinity) and β-actin (1:3000, Affinity). Thereafter, the membranes were incubated at room temperature for 1 h with horseradish peroxidase-linked secondary antibodies (1:2000, Wanleibio). The protein bands were detected by using an enhanced chemiluminescence kit (Wanleibio).
3
Immunoprecipitation and Western Blot Analysis
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Cells were lysed in NETN buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl,1% NP-40,1 mM EDTA), adding proteinase inhibitor cocktail (MedChemExpress) freshly before use. The lysate was than pre-cleaned with protein A/G Sepharose beads (GE Healthcare), and specific antibodies or control IgG was added to supernatant and incubated with blocked beads at 4°C overnight. Beads with the bound immunoprecipitates were collected and washed four times with the cold NETN buffer, and then proteins were analyzed for western blot assay.
4
Immunoprecipitation Protocol for Protein Analysis
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Cells were lysed in NETN buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 1 mM EDTA), with Proteinase Inhibitor Cocktail (MedChemExpress, #HY-K0010) added freshly before use. Then precleaned protein G sepharose beads (GE Health care) were added to the resulting lysate at 4 °C. After being precleaned, specific antibodies or control IgG was added to the supernatant, which was incubated with fetal bovine serum–blocked beads for at least 20 h at 4 °C. Beads with the bound immunoprecipitates were collected and subjected to four time washes with the cold NETN. The final immunoprecipitates were extracted for Western Blot assay.
5
Immunoprecipitation Assay Protocol
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Cells were lysed in NETN buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 1 mM EDTA), with Proteinase Inhibitor Cocktail (MedChemExpress, #HY-K0010) added freshly before use. After being precleaned by protein G sepharose beads 4 Fast Flow (GE Healthcare, #17061802), specific antibodies or control IgG was added to the supernatant, which was incubated with FBS blocked beads for at least 20 h at 4°C. Beads with the bound immunoprecipitates were collected following four washes with cold NETN. The subsequent immunoprecipitates were extracted for WB assay.
6
Immunoprecipitation of THUMPD3-HA and TRMT112-Flag
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For immunoprecipitation, HEK293T cells were transfected with pcDNA3.1(+)-THUMPD3-HA or pcDNA3.1(+)-TRMT112-Flag. Lipofectamine 2000 was used for transfection according to the manufacturer′s protocol. After transfection for 36 h, the cells were washed three times with ice-cold PBS and then lysed with 1 ml of ice-cold lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.25% deoxycholic acid) supplemented with a Proteinase Inhibitor Cocktail (MedChemExpress). The supernatant was collected by centrifugation at 12 000 rpm for 20 min. Subsequently, the supernatant was incubated with the anti-HA magnetic beads or anti-Flag magnetic beads with gentle agitation for 6 h. Recovered immunoprecipitation complexes were washed three times with ice-cold lysis buffer. All procedures were performed at 4°C. The immunoprecipitation complexes were eluted by incubating with protein loading buffer (10 mM Tris–HCl (pH 6.8), 2% sodium dodecyl sulfate, 0.1% bromophenol blue, 10% glycerol and 100 mM dithiothreitol (DTT)).
7
Immunoprecipitation and Western Blot Analysis
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Cells were lysed in NETN buffer (50 mmol/L Tris‐HCl pH 8.0, 150 mmol/L NaCl, 1% NP‐40, 1 mmol/L EDTA) with Proteinase Inhibitor Cocktail (MedChemExpress) added before use. The resulting lysate was then precleaned using 25 mL protein G Sepharose beads (GE Healthcare) at 4°C. Immunoprecipitation mixtures, including protein lysates, blocked protein G Sepharose, and the indicated Abs or IgG control derived from the same species as the indicated Ab, were incubated on a rotating wheel at 4°C overnight. Sepharose beads with the bound immunoprecipitates were collected and subjected to 4 washes with the cold NETN buffer and then analyzed by western blot assay.
8
Western Blot Protocol for Protein Analysis
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Cells were lysed in radioimmunoprecipitation lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate) containing proteinase inhibitor cocktail (MedChemExpress, Monmouth Junction, NJ, USA). The protein samples were electrophoresed through 10% SDS polyacrylamide gels and transferred onto polyvinyl difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin at room temperature for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight, and then incubated with secondary antibodies at room temperature for 1 h. Immunoreactivity was detected with enhanced chemiluminescent autoradiography (Millipore). Chemiluminescence was determined using the AI600 System (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The antibody against GAPDH (60004-1-Ig, dilution 1:1000) was purchased from Proteintech. Antibodies against HSPA8 (8444, dilution 1:1000) and DEK (29812, dilution 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA).
9
Western Blot Analysis of SFXN4, Cyclin D1, and MMP2
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Forty-eight hours after siRNA transfection, RIPA lysis buffer (Servicebio Technology, China) containing Proteinase Inhibitor Cocktail (MedChemExpress, NJ, USA) was used to lyse Huh7 and MHCC97H cells. The BCA kit (Servicebio) was applied to quantify concentration of protein. After electrophoresis, 30 μg of total proteins were separated by SDS-PAGE, then, the proteins were transferred to PVDF membranes (Millipore, USA), and the membranes were immersed in 5% skimmed milk for blocking, and then incubated individually with specific primary antibodies [the internal control α-tubulin polyclonal antibody (Proteintech, China; 1:5000), SFXN4 antibody (Affinity, USA; 1:1000), cyclin D1 antibody (Proteintech, China; 1:2000), MMP2 (Boster, China; 1:200)]. After incubation at 4 ℃ overnight, the PVDF membranes were washed and then immersed in HRP-conjugated secondary antibody for 1 h. An ECL kit (NCMBIO, China) was applied for visualization of protein bands. The density of the bands was quantified by ImageJ software (NIH, USA).
10
Antibody-based Protein Immunoprecipitation
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Cells were lysed in NETN buffer (50 mM Tris-HCl [pH = 8.0], 150 mM NaCl, 1% NP-40, 1 mM EDTA), with Proteinase Inhibitor Cocktail (MedChemExpress, #HY-K0010) added before use. After the resulting lysate was precleaned by protein G sepharose beads 4 Fast Flow (GE Healthcare, #17061802), specific antibodies or control IgG was added to the supernatant, which was incubated with FBS blocked beads on a rotating wheel at 4 °C overnight. Beads with the bound immunoprecipitates were collected following four washes with cold NETN and then the immunoprecipitates were analyzed by WB assay.
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