FHC, SW480, and SW620 cells were lysed using RIPA buffer (Beyotime, Jiangsu, China). Lysates were mixed with loading buffer (Thermo Fisher) and boiled in boiling water for 10 min. The samples were loaded on 12% bis-tris-acrylamide gel (Thermo Fisher) and protein bands were transferred onto polyvinylidene fluoride (Millipore, Bradford, MA, USA). After that, the bands were immersed in 5% nonfat milk (Solarbio, Beijing, China) at 4°C for 4 h. The membranes were incubated with anti-MSL1 (1:1000; Abcam, Cambridge, UK) and anti-β-actin (1:10000; Abcam) overnight at 4°C. And the membranes were washed with TBST (Solarbio). The secondary antibody marked with horseradish peroxidase (HRP) (1:5000; Abcam) was used to incubate the membranes at 37°C for 2 h. Protein bands were presented with enhanced chemiluminescence (Millipore). β-actin was employed as a control.
Nonfat milk
Manufactured by Solarbio
Sourced in China, United States
5% nonfat milk is a laboratory reagent used as a blocking agent in various immunoassays and blotting techniques. It is a solution containing 5% dry nonfat milk solids in water.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (6 mentions)
Bis tris acrylamide gel, by Thermo Fisher Scientific (5 mentions)
Loading buffer, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (3 mentions)
Ecl kit, by Beyotime (3 mentions)
Ab205718, by Abcam (3 mentions)
Lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Enhanced chemiluminescence, by Keygen Biotech (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (2 mentions)
Bca kit, by Solarbio (2 mentions)
Ab181602, by Abcam (2 mentions)
Rapidstep ecl reagent, by Merck Group (2 mentions)
Ecl chemiluminescence kit, by Ipsen (2 mentions)
Bca kit, by Boster Bio (2 mentions)
24 protocols using nonfat milk
1
Western Blot Analysis of MSL1 Protein
2
Western Blot Analysis of Protein Expression
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The cells were collected after transfection, and total protein was extracted from two kinds of cells using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China). The protein concentration was estimated using the bicinchoninic acid (BCA) protein assay (Boster, China). Equal amounts of prepared protein were loaded onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for electrophoresis and then transferred onto polyvinylidene fluoride membranes. Then, we used 5% nonfat milk (Solarbio, China) to block the membranes at room temperature for 1 h. Subsequently, the membranes were incubated with anti-UBE2C (1:2000; ab125002), anti-GAPDH (1:600; CST5174), anti-p-PTEN (1:2000; CST9551), anti-p-AKT (1:1000; CST4060), anti-AKT (1:1000; CST4691), anti-p-mTOR (1:1000; CST5536), anti-mTOR (1:1000; CST2972), or anti-HIF-1α (1:1000; CST36169) at 4°C overnight. Subsequently, the membranes were briefly rinsed with tris buffered saline with tween (TBST) three times (10 min each), incubated with an anti-rabbit secondary antibody for 1 h (1:2000, CST7074), and then washed with TBST three times (10 min each). Finally, the protein bands were visualized using an electrochemiluminescence (ECL) kit (Beyotime, China).
3
Western Blot Analysis of Apoptosis Regulators
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CRC tissues and cells were treated with lysis buffer (Beyotime) to prepare protein samples. Protein concentration was measured using bicinchoninic acid assay (BCA) kit (Beyotime). And 20 μg protein was loaded on 12% bis-tris-acrylamide gel (Thermo Fisher). Then, protein bands were transferred onto polyvinylidene fluoride (Millipore) and immersed in 5% nonfat milk (Solarbio, Beijing, China) at 4 °C for 4 h. After incubated with anti-B cell lymphoma-2 (anti-Bcl-2) (1:1000; CST, Boston, MA, USA), anti-BCL2-associated x protein (anti-Bax) (1:1000; CST), anti-GRIK3 (1:1000; Abcam, Cambridge, UK), and anti-β-Actin (1:1000; CST) at 4 °C overnight, the membranes were incubated with the secondary antibody labeled with horseradish peroxidase (1:2000; CST) at 37 °C for 2 h. The protein bands were developed using a Clarity™ ECL Substrate Kit (Bio-Rad, Shanghai, China). β-Actin was chosen as a control.
4
Western Blot Analysis of Protein Expression
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Cells were homogenized in RIPA lysis buffer (Solarbio, Beijing, China) containing protease inhibitor cocktail (Roche, Mannheim, Germany) on ice. After the mixture was centrifuged at 12000g for 10 min at 4°C, the supernatant was collected. The protein concentration was measured using BCA Protein Assay Kit (Beyotime, Beijing, China) according to the manufacturer’s protocol. Proteins were separated by SDS/PAGE and then transferred on to PVDF or NC membranes (Solarbio, Beijing, China). The membranes were blocked in TBST (TBS with 0.1% Tween 20, pH: 7.2) containing 5% non-fat milk (Solarbio, Beijing, China) for 1 h, then incubated with primary antibodies (Table 2 ), followed by HRP–conjugated secondary antibodies (Table 3 ) at room temperature. Proteins were detected using the chemiluminescent kit (TransGen Biotech, Beijing, China).
5
Western Blot Analysis of BDNF Protein
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OS tissues and cells were lysed with RIPA buffer (Beyotime). Lysate was boiled in boiling water for 8 min and suspended in loading buffer (Solarbio, Beijing, China). Protein sample was loaded by 12% sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime). Protein bands were transduced onto nitrocellulose membranes (GE Healthcare, Westborough, MA, USA) and incubated in 5% nonfat milk (Solarbio) at 4 °C for 5 h. After that, membranes were incubated with anti-BDNF (1:1000; CST, Boston, MA, USA) and anti-GAPDH (1:1000; CST) at 4 °C overnight. The membranes were incubated with secondary antibody marked horseradish peroxidase (1:2000; CST) at 37 °C for 2 h. Protein bands were visualized under enhanced chemiluminescence (KeyGen, Nanjing, China). GAPDH was employed as a reference.
6
Protein Extraction and Western Blot Analysis
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Total protein was extracted by RIPA lysis buffer mixed with PMSF (Solarbio). A BCA protein assay kit was used to determine the protein concentration (Beyotime, P0009, Shanghai, China). Proteins (20 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (8% for CD206, CD163, CD68, HIF1α and LATS1, 10% for TSG101, CD63, Calnexin, pYAP1, YAP1 and GAPDH, and 12% for CD9 and VHL) and transferred to a PVDF membrane (Millipore Corporation, MA, USA). After membranes were blocked with 5% nonfat milk (Solarbio) at room temperature, the following primary antibodies were used to incubate membranes overnight at 4 °C (anti-CD68, 1:500, 28058-1-AP, Proteintech; anti-CD206, 1:600, 60143-1-Ig, Proteintech; anti-CD163, 1:100, sc-20066, SCBT, CA, USA; anti-VHL40, 1:200, sc-135657, SCBT; anti-LATS1, 1:5000, 17049-1-AP, Proteintech; anti-YAP1, 1:4000, 13584-1-AP, Proteintech; anti-pYAP1, 1:4000, T55743, Abmart, Shanghai, China; anti-HIF-1α, 1:500, ab308433, Abcam; anti-GAPDH, 1:8000, ab8245, Abcam). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:8000, ab205719/ab205718, Abcam) for 1 h, and the protein bands were detected using an ECL detection system (1705061, Bio-Rad).
7
Western Blot Analysis of PTEN Protein
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Proteins were collected from HCMs cells through protein lysis buffer (Solarbio) and quantified by a BCA kit (Solarbio). Proteins (80 μg) were electrophoresed via 10% SDS‐PAGE gel and transferred to the NC membrane (Millipore). Then, the membrane was blocked by 5% nonfat milk (Solarbio) for 1 h at room temperature, and then incubated with the corresponding primary antibodies overnight at 4°C. The next day, membranes were washed 3 times with 0.1% TBST buffer and then incubated with HRP‐conjugated secondary antibodies for 1 h at room temperature. Primary antibodies were shown as follows: PTEN (1:1000; ab31392, Abcam), β‐actin (1:5000; ab8227, Abcam). The blot signals were detected by the ECL kit (Pierce), and the intensity was quantified via Image J 6.0 (Bio‐Rad).
8
Western Blot Analysis of Protein Signaling
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Cells were lysed with RIPA lysis buffer (Beyotime), and protein concentrations were determined by BCA. The samples were separated by SDS-PAGE. Gel proteins were transferred to PVDF membranes (Millipore), sequentially blocked with 5% nonfat milk (Solarbio), and then incubated with primary antibodies overnight at 4 °C. PVDF membranes were further incubated with secondary antibodies, and chemiluminescent detection was performed using the Western Blotting Detection Kit ECL (Human IgG) (Solarbio). Primary antibodies used for western blot included p-Aurora-A (host species: Rat, #3079, CST), Aurora-A (host species: Rat, #ab52973, Abcam), GAPDH (host species: Rabbit, #ab181602, Abcam), PFKFB3 (host species: Rat, # ab181861, Abcam), p-PFKFB3 (host species: Rat, # ab202291, Abcam), p-ERK (host species: Rat, #4370, CST), ERK (host species: Rat, #4695, CST), p-AKT (host species: Rat, #a4060, CST), and AKT (host species: Rat, #4685, CST). GST (host species: Rat, # ab252882, Abcam). ImageJ software was performed to quantify the band intensities of Western blot figures.
9
Western Blot Analysis of Ovarian Cancer
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Ovarian cancer tissues and cells were lysed with lysis buffer (Beyotime). The lysates were boiled and mixed with loading buffer (Thermo Fisher). Lysates were loaded onto 12% bis-tris-acrylamide gels (Thermo Fisher), and proteins were transferred onto polyvinylidene fluoride (Millipore). Membranes were immersed in 5% nonfat milk (Solarbio) at 4 °C for 4 h. Following that, these membranes were incubated with the antibodies against GAPDH (ab128915; 1:20000; Abcam) or the long isoform of CBX2 (ab182620; 1:500; Abcam) overnight at 4 °C, respectively. Secondary antibody labeled with horseradish peroxidase (ab205718; 1:5000; Abcam) was used to incubate membranes at 37 °C for 2 h. Protein bands were visualized using enhanced chemiluminescence system (Pierce, Rockford, IL, USA). GAPDH was employed as a reference.
10
Protein Expression Analysis in A549 and H1299 Cells
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A549 and H1299 cells were harvested 72 h after transfection and lysed with cold RIPA lysis buffer (Beyotime) containing PMSF for 20 min on ice. After centrifugation at 14,000 x g for 10 min, the supernatant was decanted into a new tube. Subsequently, we used a BCA protein assay kit (Solarbio) to measure the protein concentration. 40 µg of protein and 5ul of protein marker were resolved by 7% SDS-PAGE and the gel contents electrotransferred onto PVDF membranes. After incubation in 5% non-fat milk (Solarbio) for 2 h, the membrane and primary antibodies were incubated, with rocking, overnight at 4 °C. After TBST washes, PVDF membranes and corresponding secondary antibodies (ABMARK) were incubated at 37 °C for 2 h. Finally, we used an ECL kit (Beyotime) to visualize protein bands on the membrane. Assessment of protein abundance was determined using Image J software.
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