Protein expressions of FOXP3 and RoR-γt in mice kidney tissues were quantified using Western blot analysis. Mice kidney tissues were lyzed by NP-40 Lysis Solution (N8032; Solarbio) which added with protease inhibitor mixture (P6730; Solarbio) and PMSF (P0100; Solarbio) to obtain the total protein in tissues. Then, the concentration of total protein was evaluated using a BCA protein assay kit (PC0020; Solarbio). After the protein was denatured by mixing with loading buffer (P1040; Solarbio) and 100 °C heating for 5 min, the protein was separated by the SDS-PAGE gel (P1200, Solarbio) and transferred onto PVDF membrane (YA1701, Solarbio). Then, the PVDF membrane was incubated with western blocking buffer (SW3010; Solarbio) for 2 h followed by incubating FOXP3 antibody (ab215206; 1:1000, 47 kDa, Abcam, Cambridge, UK), RoR-γt antibody (ab113434; 1:2000, 55 kDa, Abcam), and GAPDH antibody (ab8245; 1:10,000, 36 kDa, Abcam) for 16 h at 4 °C. On the second day, membrane was further incubated with relative goat-anti rabbit IgG (ab6721; 1:20,000, Abcam), goat-anti mouse IgG (ab6789; 1:10,000, Abcam), or donkey-anti goat IgG (ab6885; 1:10,000, Abcam) antibody for 2 h. At last, after the membrane was added with ECL Luminescent Liquid (M41129, MERYER), the protein signaling on the membrane was examined by Image Lab 3.0 detector (Bio-Rad, Hercules, California, USA).
P1200
Manufactured by Solarbio
Sourced in China, United States
The P1200 is a single-channel electronic pipette with a volume range of 100 to 1200 μL. It is designed for accurate and precise liquid handling in laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (5 mentions)
Ab6721, by Abcam (4 mentions)
R0010, by Solarbio (4 mentions)
Ipvh00010, by Merck Group (4 mentions)
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Ya1701, by Solarbio (2 mentions)
P1040, by Solarbio (2 mentions)
Iseq00010, by Merck Group (2 mentions)
Ab40772, by Abcam (2 mentions)
Ab76003, by Abcam (2 mentions)
Ab76011, by Abcam (2 mentions)
Ab276131, by Abcam (2 mentions)
Ab92536, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab92547, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Zd409, by ZOMANBIO (2 mentions)
Total protein extraction kit, by Solarbio (2 mentions)
Ab32503, by Abcam (2 mentions)
29 protocols using p1200
1
Quantification of FOXP3 and RoR-γt Protein Expressions in Mice Kidneys
2
Western Blot Protein Analysis Protocol
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The total protein was lysed from cells in radio immuno precipitation assay buffer (R0010, Solarbio) with protease and phosphatase inhibitor (P1045, Beyotime, 1:50) for 30 min at 4°C, and then quantified with a BCA kit (PC0020, Solarbio). ColorMixed Protein Marker (PR1920, Solarbio) served as a protein size marker. Then, protein (20 μg) was isolated by 6–10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels (P1200, Solarbio) and transferred onto immobilon-P polyvinylidene fluoride membranes (YA1701, Solarbio). Following incubation with 5% bovine serum albumin (SW3015, Solarbio) for 1 h at room temperature, the membranes were treated with primary antibodies (shown in Table 2 ) at 4℃ overnight, and then corresponding secondary antibody (shown in Table 2 ) for 2 h at room temperature. Visualization was achieved with ECL Western Blotting Substrate (PE0010, Solarbio). The intensity of protein band was determined by Quantity One Analysis Software (version 4.62; Bio-Rad Laboratories, Inc.).
3
Liver Protein Expression Analysis
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Total protein was extracted from the liver tissue and HepG2 cells, and the total protein concentration was determined using the BCA method. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (P1200, Solarbio, China) and transferred to polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Millipore, USA). The membranes were blocked with 5% nonfat milk (P0216-300g, Solarbio, China) containing 0.1% Tween® 20 (T8220, Solarbio, China). After washing with 0.1% Tris-buffered saline with Tween® 20 (TBST), the membranes were incubated with specific antibodies overnight. After being washed in TBST, the membranes were incubated with anti-rabbit immunoglobulin G (IgG) and horseradish peroxidase (HRP)-linked antibody diluted at 1:2000 (7074S, CST, USA) for 1-h. The protein expression was detected by a GelView system (6000plus, BLT, China), and immunoreactive bands were analyzed by ImageJ (ImageJ 1.45s, USA). The specific antibodies were against PPARα (ab24509, 1:500, Abcam, China), CPT1A (bs-2470r, 1:2000, Bioss, China), CD36 (bs-1100r, 1:2000, Bioss, China), and FABP1 (ab153924, 1:2000, Abcam, UK). β-actin (4970T, 1:3000, CST, USA) was used as an internal control.
4
Western Blot Analysis of Proteins
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All proteins from cells or tumors were resolved by SDS-PAGE gel (P1200, Solarbio, Beijing, China) and transferred onto PVDF membranes (LC2002, Thermo, Waltham, US). Then, the membranes were blocked in 5% fat-free milk in TBST for 1 hour at room temperature and then washed, and primary antibodies were incubated at 4°C overnight. After being washed, the PVDF was incubated with horseradish peroxidase-conjugated secondary antibodies. The bands were visualized by ECL reagent (32106, Thermo). Antibodies are as follows: KIF11 (1 : 1000 dilution, ab61199, Abcam, Cambridge, UK); anti-β-actin (1 : 1000 dilution, ab5694, Abcam, Cambridge, UK).
5
Western Blot Analysis of TTK, Ki67, and PCNA
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Cells were washed three times with cold PBS and lysed in RIPA (89900, Thermo, Waltham, USA) containing 1% PMSF (36978, Thermo, Waltham, USA), and then the cells were incubated on a shaking table in 4°C for 30 min. The lysate was centrifuged at 12000 r/min for 30 min at 4°C. 30 µg of protein, the concentration determined using Bradford assay (P0006, Beyotime, Shanghai, China), was electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (P1200, Solarbio, Beijing, China) at 90 V, and then transferred to PVDF (ARKEMA, Colombes, France) membranes at 250 mA for 130 min. The membranes were blocked with 5% non-fat milk for 1 h at room temperature after being washed three times with tris-buffered saline and Tween (TBST). Then, the PVDF was incubated overnight with primary antibodies on a shaking table at 4°C. Primary antibodies were: TTK (ab219068, 1 : 1000, Abcam, Cambridge, UK), β-actin (1 : 5000, KM9001T, Sungene, Tianjin, China), ki67 and proliferating cell nuclear antigen (PCNA) (1 : 1000, 9449T, 2586S, Cell Signaling Technology, Danvers, UK). Then, the second antibodies (1 : 10000, LK2003, Sungene, Tianjin, China) were incubated for 1 h on a shaking table at 37°C after washing three times with TBST. Finally, ECL reagents (35050, Thermo, Waltham, USA) were added to display strips and take pictures using an image capture system.
6
Western Blot Analysis of Epithelial-Mesenchymal Markers
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After being separated by SDS-PAGE (P1200, Solarbio, China), total proteins were transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Afterwards, membranes were incubated by primary antibodies at 4 °C overnight. Then the blots were then incubated with secondary antibody for 1 h at dark room after washed with PBS for three times. The primary antibodies were as follows: ZO-1 (ab276131, Abcam), E-cadherin (ab40772, Abcam), N-cadherin (ab76011, Abcam), Vimentin (ab92547, Abcam), Fibronectin, NAP1L2 (K21628-RNX, Biolab), HNRNPC, METTL14 (ab220030, Abcam), YY1 (ab109237, Abcam), MMP2 (ab92536, Abcam), MMP9 (ab76003, Abcam) and β-actin (ab8226, Abcam). Protein quantification was conducted by Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerican MA, USA). The assay was independently carried out in triplicate.
7
Protein Expression Analysis by Western Blot
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Cells and tissues were lysed using a high-efficiency RIPA lysis buffer (Solarbio). Protein separation was performed by SDS-PAGE (P1200, Solarbio). Following membrane transfer, primary antibodies were applied. These primary antibodies included anti-PD-L1 (ab282458, 1:1000, Abcam), anti-CNDP1 (ab155315,1:500, Abcam) and β-actin (ab8227, 1:1000, Abcam). Secondary antibodies were goat anti-rabbit IgG (ab6721, 1:2000, Abcam). Chemiluminescence was performed using the ECL chromogenic reagent, and bands were observed using an automated chemiluminescence analyser (PerkinElmer, Waltham, MA, USA).
8
Protein Extraction and Western Blot Analysis
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Protein extraction from HCC cells was conducted using protein lysis buffer (ZD409, ZOMANBIO, Beijing, China) and a Total Protein Extraction Kit (BC3711, Solarbio, Beijing, China). The obtained proteins were separated via SDS–PAGE (P1200, Solarbio) and then transferred onto PVDF membranes (T2234, Thermo Fisher). Following blocking with 5% skimmed milk, the membranes were incubated with primary antibodies. Following this, the membranes underwent incubation with secondary antibodies for 1 h. Protein levels were determined using an enhanced chemiluminescence detection system, with β‐actin serving as the internal reference.
9
Western Blot Analysis of Aortic Proteins
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Aorta tissues were homogenized in radioimmunoprecipitation assay (RIPA; Solarbio, R0010, Beijing) lysis buffer. Equal amounts of protein (40 μg) were extracted from the total aorta and separated by 10% SDS–polyacrylamide gel electrophoresis (Solarbio, P1200, Beijing). The proteins were then transferred to polyvinylidene fluoride (PVDF; Millipore, ISEQ00010, USA) membranes and blocked with 5% skim milk powder (BD, 232100). The following primary antibodies were incubated with PVDF membranes at 4°C overnight: NF-κB antibody (1 : 2000, GeneTex, GTX102090), anti-thioredoxin interacting protein (TXNIP) antibody (EPR14774) (1 : 1000, Abcam, ab188865), NLRP3 antibody (1 : 1000, GeneTex, GTX64347), anti-ASC antibody (1 : 200, Abcam, ab175449), caspase-1 antibody (14F468) (1 : 1000, GeneTex, GTX14367), IL-1β antibody (1 : 2000, GeneTex, GTX55675), and β-actin antibody (1 : 5000, GeneTex, GTX109639). The PVDF membranes were then extensively rinsed and incubated at room temperature with secondary antibodies (1 : 5000, Proteintech, SA00001-1, SA00001-2, Wuhan) for 1 h. A biomolecular imager (Fuji, LAS-4000, MINI, Japan) and QualityOne were used to scan and quantitate the membranes. All experiments were repeated 3 times.
10
Protein Extraction and Western Blot Analysis
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The total protein of each group was extracted by a protein extraction kit (KGP250, KeyGEN, China) and quantified by a protein quantification kit (KGP902, KeyGEN, China). The protein sample size was 20 µg. The total protein was obtained by SDS‒PAGE (P1200, Solarbio, China), and the protein was transferred to solid-phase carrier PVDF membrane (IPFL00010, Millipore, Germany), and the primary antibody was added: anti-RAGE (ab216329, abcam, UK), anti-HMGB1 (6893, CST, USA), anti-IL-17RA (bs-2606R, Bioss, China), anti-TAK1 (ab109526, abcam, UK), anti-Phospho-TAK1 (bs-5435R, Bioss, China), anti-β-actin (bs-0061R, Bioss, China), incubated overnight at 4 °C, added secondary antibody: Goat anti-rabbit (bs-0295G, Bioss, China), Goat anti-mouse (bs-0296G, Bioss, China), incubated at room temperature for 40 min and added substrate (WBKLS0100, Millipore, Germany), developed by chemiluminescent image (A300, Azrue, USA).
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