Detailed information about the Western blot analysis was previously described (Liang et al., 2022a (link)). The antibodies used are as follows: β-actin antibody (AF7018, Affinity), KIF20A antibody (AF7664, Affinity), Goat Anti-Rabbit IgG (H + L) HRP (S0001, Affinity) and Goat Anti-Mouse IgG (H + L) HRP (S0002, Affinity).
S0002
Manufactured by Affinity Biosciences
Sourced in United States
The S0002 is a high-performance laboratory centrifuge. It is capable of separating and isolating various sample components based on differences in their density and size. The centrifuge operates at variable speeds to accommodate a range of sample types and volumes.
Automatically generated - may contain errors
Lab products found in correlation
S0001, by Affinity Biosciences (7 mentions)
Af7018, by Affinity Biosciences (4 mentions)
Goat anti mouse igg h l hrp, by Affinity Biosciences (3 mentions)
Goat anti rabbit igg h l hrp, by Affinity Biosciences (2 mentions)
Af0199, by Affinity Biosciences (2 mentions)
Af7017, by Affinity Biosciences (2 mentions)
β actin antibody, by Affinity Biosciences (1 mentions)
P0013, by Beyotime (1 mentions)
Ps107, by Ipsen (1 mentions)
Ecl chemiluminescent substrate kit, by Yeasen (1 mentions)
Bca protein colorimetric assay kit, by Elabscience (1 mentions)
Gs 800 calibrated densitometer, by Bio-Rad (1 mentions)
Protein extraction kit, by Keygen Biotech (1 mentions)
Enhance chemiluminescence, by Merck Group (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Ecl chemiluminescence detection kit, by Biosharp (1 mentions)
Ripa buffer, by CWBIO (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
12 protocols using s0002
1
Detailed Western Blot Protocol
2
Western Blot and Immunoprecipitation Protocol
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Cell lysis buffer for Western and IP (P0013, Beyotime) was used to lyse HGC27 and AGS cells after they had been exposed to various doses of NTP for 24 h. The BCA protein Colorimetric Assay Kit (E-BC-K318-M, Elabscience) was subsequently utilized to determine the total protein. After the protein was separated by electrophoresis, the membrane was transferred using polyvinylidene difluoride (PVDF) at an ice bath temperature. Five percent skim milk was used to block the portions. In a 4 °C shaker, the matching antibodies were incubated overnight. After rinsing with Tris-buffered saline and Tween (TBST), the film was treated with secondary antibodies (S0002, Affinity) for 1.5 h at room temperature. Finally, the imaging system was used to build the improved ECL chemiluminescent substrate kit (36222, Yeasen). For some of the PVDF membranes, Western blot fast stripping buffer (PS107, Epizyme) was used. After completing Western blot luminescence detection, the PVDF membranes were rinsed with TBST for 5 min. Add 10 mL of fast stripping buffer to cover the membrane and rinse for 20 min. The stripping solution was removed, and TBST was added to rinse the PVDF membrane for 5 min. The membrane was sealed with skim milk powder for 2 h, the PVDF membrane was washed with TBST, and then the other antibodies were added again. The gray value was analyzed and calculated using ImageJ software.
3
Western Blot Analysis of Cellular Proteins
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Total cellular protein was extracted with the protein extraction kit (KeyGen Biotech, Nanjing, China). The modified Lowry protein assay kit (Pierce, Rockford, IL, United States) was used to quantify protein. Equal amounts of protein were analyzed using 10% SDS–PAGE, and transferred to a PVDF membrane. PVDF membranes were incubated with primary rabbit anti-LC3B (CST, 3868S,1:1200), primary mouse anti-cathepsin B (ab58802,1:1000), rabbit anti-P53 (ab131442,1:1000), rabbit anti-p21Cip1p (CST,2947S,1:1000), rabbit anti-p16INKa (CST,80772S,1:1000), rabbit anti-P62 (CST,16177S,1:1000), and anti-GAPDH (ab181602,1:2000) incubated overnight at 4°C. Following subsequent incubation with anti-rabbit HRP-conjugated secondary antibody (Affinity, S0001, 1:2000) or anti-mouse HRP-conjugated secondary antibody (Affinity, S0002, 1:2000), blots were visualized with enhance chemiluminescence (Millipore, Billerica, MA, United States). Bands on X-ray film were scanned with GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA, United States). The intensity of bands was quantified with Quantity one software. All values were normalized to the corresponding GAPDH.
4
Protein Extraction and Western Blot Analysis
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Total protein from samples was extracted using RIPA buffer (Cwbio, Beijing, China). The protein concentration was then quantified using the BCA Protein Assay Kit (Cwbio, Beijing, China) and diluted accordingly. Equal amounts of protein were separated via SDS-PAGE with 8% or 10% polyacrylamide gels, depending on the molecular weight of the protein. The separated proteins were then transferred to a 0.45 μm PVDF membrane (Millipore,St. Louis, MO, USA) via electrophoresis, followed by blocking with 5% BSA at room temperature for 2 h. Primary antibodies for each protein were diluted in 5% BSA as follows: Fibronectin (1:1000, 26836S, CST), Col-III (1:1000, 66887S, CST), VASH-1 (1:1000, sc-365541, Santa Cruz), p-Smad3 (1:1000, 9520S, CST), Smad3 (1:1000, 9513S, CST), β-actin (1:1000, #AF7018, Affinity), and EGFP (1:5000, SRP15324, Saier); the secondary antibodies used were anti-rabbit (1:40,000, S0001, Affinity) and anti-mouse (1:40,000, S0002, Affinity). All primary antibodies were incubated overnight at 4 °C, followed by 1.5 h of incubation at room temperature with the corresponding secondary antibodies. Detection was performed using the ECL chemiluminescence detection kit (Biosharp, Guangzhou, China). Image collection and quantitative analysis were conducted using ImageJ software v1.8.0.
5
Protein Extraction and Western Blot Analysis
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Protein samples were extracted from cells or tissues with RIPA lysis buffer (Beyotime, P0013B, Shanghai, CN). Protein concentration was detected with a BCA Protein Assay Kit (Beyotime, P0012S, Shanghai, CN). Western blotting was prepared according to previous study[27 (link)] Primary antibodies against GAPDH (1:1000, AB-P-R 001, Hangzhou Xianzhi Biological Co., Ltd., Hangzhou, CN), pepsin (1:1000, DF8591, Affinity Biosciences, Melbourne, AUS), Bax (1:1000, 50599-2-Ig, San Ying Biotechnology, Wuhan, CN), Bcl-2 (1:1000, 26593-1-AP, San Ying Biotechnology, Wuhan, CN), caspase3 (1:1000, Ab184787, Abcam, Cambridge, UK), GLUT1 (1:1000, AF6731, Affinity Biosciences,Melbourne, AUS), MCT4 (1:1000, DF4182, Affinity Biosciences, Melbourne, AUS), and HK-II (1:1000, DF6176, Affinity Biosciences, Melbourne, AUS) were used to detect protein levels of these molecules. The next day, the membrane was soaked with secondary antibodies [HRP-labeled sheep anti-rabbit secondary antibody, 1:10000, BA1054, Wuhan Bode Bioengineering Co., Ltd., Wuhan, CN; goat anti-mouse IgG (H + L) HRP, 1:5000, S0002, Affinity Biosciences, Melbourne, AUS]. The membrane was subsequently scanned (Canon, K10486, Jap), and grayscale value of protein expression was analyzed by BandScan (Glyko, USA).
6
Western Blot Analysis of Inflammatory Markers
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All samples which had 40 μg total protein were loaded on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated overnight at 4°C with rabbit anti‐FKN (DF12376, 1:500, Affinity), rabbit anti‐iNOS (AF0199, 1:500, Affinity), rabbit anti‐Wnt‐4 (DF9040, 1:500, Affinity), rabbit anti‐β‐catenin (AF6266, 1:500, Affinity), rabbit anti‐c‐Myc (AF0358, 1:500, Affinity), rabbit anti‐CyclinD1 (AF0931, 1:500, Affinity), rabbit anti‐TNF‐α (DF7014, 1:500, Affinity), rabbit anti‐IL‐10 (DF0175, 1:500, Affinity), anti‐ARG‐1 (DF6657, 1:500, Affinity), rabbit monoclonal FKN antibody (ab25091, 1:1000, Abcam), rabbit monoclonal anti‐Wnt‐4(ab262696, 1:1000, Abcam), rabbit monoclonal anti‐β‐catenin (ab6302, 1:1000, Abcam), mouse anti‐β‐actin (AF7018, 1:1000, Affinity), mouse anti‐β‐tubulin (AF7010, 1:10 000, Affinity) and mouse anti‐GAPDH (AF7021, 1:1000, Affinity). The membranes were then incubated with goat anti‐rabbit IgG (S0001, 1:5000, Affinity) and goat anti‐mouse IgG (S0002, 1:5000, Affinity) for 50 minutes at room temperature. Then, they were exposed to an enhanced chemiluminescence substrate (KF003, Affinity) and visualized using Tanon‐5200 (Tanon, Shanghai, China).
7
Western Blot Analysis of Cellular Signaling
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Cells and aortas were lysed in RIPA buffer (Beyotime, Guangzhou, China), and 40 µg total protein was resolved in 12% or 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membrane, which was then blocked with 5% skimmed milk. Primary antibodies against iNOS (1:2,000, AF0199, Affinity, USA), COX-2 (1:2,000, AF7003, Affinity), CHOP (1:1,000, 2895, CST, USA), BCL-2 (1:1,000, YT0470, ImmunoWay, USA), BAX (1:1,000, YT0455, ImmunoWay), cleaved Caspase 3 (1:1,000, AF7022, Affinity), cleaved Caspase 8 (1:1,000, AF5267, Affinity), TIPE2 (1:1,000, DF3326, Affinity), TLR4 (1:2,000, AF7017, Affinity), TRIF (1:2,000, DF6289, Affinity), MyD88 (1:2,000, AF5195, Affinity), AKT (1:1000, YM3618, ImmunoWay), p-AKTSer473 (1:1000, YP0006, ImmunoWay), and β-actin (1:10,000, AF7018, Affinity) were used. HRP-conjugated corresponding secondary antibodies (1:10,000, S0001 and S0002, Affinity) were used, and antigen–antibody reactions were visualized using an enhanced chemiluminescence detection kit (E411-05, Vazyme, China). ImageJ 1.48 (NIH, Maryland, USA) was used to quantify the intensities of each band. β-actin served as an internal reference.
8
Western Blot Analysis of Protein Expression
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Cells were washed twice with ice-cold PBS, then lysed in SDS lysis buffer containing 1× protease inhibitor cocktail (Roche Applied Science, Germany). The total protein concentration in the cell lysate solution was then determined via the BCA protein assay (Thermo Fisher Scientific, USA). Protein samples (20 µg) were separated by electrophoresis on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, USA). After being blocked with 5% skim milk (BD Biosciences, USA) for 1 h, the membranes were incubated with specific primary antibodies; β-actin (Affinity Biosciences, USA, T0022; 1:4,000), G3BP1 (Santa Cruz, USA, sc-98,561; 1:1,000), AR (Abcam, USA, ab108,341, 1:3,000) overnight at 4 °C. After washing with TBST, the membranes were incubated with HRP-conjugated anti-mouse (Affinity Biosciences, USA, S0002, 1:3,000) or anti-rabbit (Affinity Biosciences, USA, S0001, 1:5,000) secondary antibodies for 1 h and visualized with an ECL system (Millipore, USA).
9
Gastrocnemius Protein Extraction and Analysis
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The acquired gastrocnemius tissues were lysed with the assistance of a lysis buffer (abs9229, absin, China). Subsequently, we selected the BCA kit (BI-WB005, SBJBIO, China) to quantify the lysed protein. Thereafter, the quantified protein was electrophoresed for protein separation, which was then loaded onto the PVDF membrane (PW0034, Leagene, China). After being sealed in 5% bovine serum albumin (BL-082, SBJBIO, China) at 37°C for 60 min, the membrane underwent primary antibodies (4°C, overnight). Afterward, the bound antibodies were then exposed to anti-rabbit secondary antibody (31466, Invitrogen, USA) or anti-mouse secondary antibody (S0002, Affinity, USA) with the condition of 37°C for 60 min. Visualization of protein was conducted by applying an ECL reagent (GK10008, GlpBio, USA) on a gel imaging system (A44114, Invitrogen, USA). The primary antibodies of PGC-1α (1:1,000, ab191838), P62 (1:10,000, ab109012), LC3B (1:2,000, ab48394), AMPKα (1:5,000, ab32047), p-AMPKα (1:10,000, ab133448), Smad3 (1:1,000, ab208182), p-Smad3 (1:2,000, ab52903), and β-actin (1:5,000, ab8227) were provided by Abcam (UK).
10
Western Blot Analysis of Osteopontin
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GC and paired adjacent normal tissues were collected and lysed in RIPA buffer (R0010; Solarbio, Beijing, China). We used anti-osteopontin (1:2000 dilution; 22952-1-AP; Proteintech, Wuhan, China) and anti-GAPDH (1:10,000 dilution; AC002; ABclonal, Wuhan, China) as primary antibodies and goat anti-rabbit IgG (H + L) HRP (1:5000 dilution; S0001; Affinity Cincinnati, OH, USA) and goat anti-mouse IgG (H + L) HRP (1:5000 dilution; S0002; Affinity, Cincinnati, OH, USA) as secondary antibodies.
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