After treatment, the cells were harvested in RIPA buffer supplemented with a protease inhibitor cocktail (Sigma, Shanghai, China). The protein concentration was measured by using a BCA protein assay kit (Bestbio, Shanghai, China). Aliquots of total cell lysates (50 μg protein) were mixed with loading buffer, boiled for 5 min, and separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin or 5% de-fatted milk powder for 1 h at room temperature and then incubated overnight at 4 °C with each of the following antibodies: anti-phospho-STAT1 (SAB Signalway Antibody, College Park, MD, USA), anti-STAT1 (Proteintech, IL, USA), anti-phospho-STAT2 (SAB), anti-STAT2 (Proteintech), anti-phospho-STAT3 (SAB), anti-STAT3 (Proteintech), anti-SOCS1 (Proteintech), anti-SOCS3 (Proteintech), anti-SHP1 (Proteintech), anti-SHP2 (Proteintech), anti-GAPDH (Huabio, Hangzhou, China), anti-Jak1 (SAB), anti-phospho-Jak1 (SAB), anti-Tyk2 (Proteintech), and anti-phospho-Tyk2 (SAB). Thereafter, the membranes were incubated with an appropriate peroxidase-conjugated secondary antibody. The presence of the secondary antibody was determined by the detection of enhanced chemiluminescence solution (Amersham Biosciences, Piscataway, NJ, USA).
Anti stat1
Manufactured by Proteintech
Sourced in United States
Anti-STAT1 is a protein detection reagent used in Western blot analysis. It is a primary antibody that specifically binds to the STAT1 protein, which is a key component of the JAK-STAT signaling pathway involved in cellular responses to various cytokines and growth factors.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Huabio (2 mentions)
Anti pd l1, by Proteintech (2 mentions)
Enhanced chemiluminescence solution, by Cytiva (1 mentions)
Anti stat2, by Proteintech (1 mentions)
Anti socs1, by Proteintech (1 mentions)
Anti socs3, by Proteintech (1 mentions)
Anti shp 1, by Proteintech (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bca protein assay kit, by BestBio (1 mentions)
Anti stat3, by Proteintech (1 mentions)
Anti shp2, by Proteintech (1 mentions)
Anti occludin, by Abcam (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Anti claudin 1, by Abcam (1 mentions)
Anti zo 1, by Abcam (1 mentions)
Anti hif 1α, by Affinity Biosciences (1 mentions)
Anti nf κb p65, by Abcam (1 mentions)
Bca protein concentration assay kit, by Solarbio (1 mentions)
Electronic analytical balance, by Mettler Toledo (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
6 protocols using anti stat1
1
Western Blot Analysis of JAK-STAT Signaling
2
Inflammatory Pathways Characterization
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Microfuge 22R desktop refrigerated centrifuge (Beckman, Germany), ultra-low temperature refrigerator (Anhui Zhongke Duling Co., Ltd.), electric thermostatic water bath (Changzhou Putian Instrument Manufacturing Co., Ltd.), electronic analytical balance [Mettler-Toledo Instruments (Shanghai) Co., Ltd.], electric heating constant temperature blast drying oven (Shanghai Yuejin Medical Equipment Factory), physiological saline (Shijiazhuang Siyao Co., Ltd.), BSA24S-CW electronic analytical balance (Sartorius, Germany), paraformaldehyde fixative solution (Wuhan Sewell Biotechnology Co., Ltd.); TissueLyser-24 (Shanghai Jingxin Industrial Development Co., Ltd.), BCA protein concentration assay kit (Beijing Solarbio company), NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific, United States), Mouse IFN-γ enzyme-linked immunosorbent assay kit (Jianglai Biotechnology), Mouse TNF-α ELISA kit (Jianglai Biotechnology), Mouse IL-6 ELISA kit (Jianglai Biotechnology), Anti-Claudin 1 (Abcam, United States); Anti-Occludin (Abcam, United States), Anti-ZO1 (Abcam, United States); Anti-HIF-1α (Affinity Biosciences, United States); Anti-NF-κB p65 (Abcam, United States); Anti-STAT1 (Proteintech).
3
Immunoblotting Analysis of Protein Expression
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Extracts of cells or tissue lysed with RIPA buffer were cleared by centrifugation. Lysates (30 µg of protein) were subjected to SDS-PAGE, and the separated bands were transferred to a polyvinylidene difluoride membrane (Millipore) that was then probed with various antibodies. The following antibodies were used: anti-PD-L1 (1:1000; Cat No. 66248-1-Ig; Proteintech), anti-IFN-γ (1:200; Cat No. bs-0480R, Bioss), anti-STAT1 (1:2000; Cat No. 10144-2-AP; Proteintech), anti-pSTAT1 (Tyr701) (1:500; Cat No. AF3300, Affinity), anti-Ac-lys (1:500; Cat No. A2391, ABclonal), anti- NF-κB p65 (1:1000; Cat No. ET1603-12, Huabio), anti-HDAC6 (1:1000; Cat No. ab133493, Abcam), anti-Histone H3 (1:1000; Cat No. A2348, ABclonal), and anti-GAPDH (1:1000; Cat No. ET1601-4, Huabio). Cytoplasmic and nuclear fractions were isolated with nuclear and cytoplasmic protein extraction kits (Beyotime).
4
Western Blot Profiling of HPV Biomarkers
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Cultured cells were lysed with lysis buffer. Equal amounts of protein were run on 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Millipore). After blocking with 5% milk in TBST, membranes were incubated with primary antibodies overnight. The following antibodies were used: anti-HPVE6 (1:1000, Arigo), anti-HPVE7(1:1000, Bioss), (anti-CXCL10 (1:1,000, Abcam), anti-CXCR3 (1:1,000, Boster), anti-PDL1 (1:2,000, proteintech), anti-STAT1 (1:1,000, proteintech), anti-pSTAT1 (1:1,000, Abcam), anti-JAK1 (1:2,000, proteintech), anti-GAPDH (1:6,000, Yeasen) and anti-tublin (1:3,000, Yeasen). Membranes were then incubated with the rabbit peroxidase-conjugated secondary antibody (1:10,000, Abclonal). The blots were detected by sensitive chemiluminescence liquid analysis (Yeasen) and Biorad software was used to capture the images.
5
Western Blot Analysis of Protein Expression
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The whole proteins from jejunums of mice and FHs74Int cells were extracted with RIPA lysis buffer (Beyotime Biotechnology). BCA protein assay kit (Beyotime Biotechnology) was used to determine the concentration of protein. Proteins were separated with SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany) followed by blocking with 5% skim milk (BD/Difco, Sparks, MD, USA). The various primary antibodies used in the experiments were as follows: anti-GPX4 (1:1000; Protein Tech Group, Wuhan, China), anti-ACSL4 (1:1000; ABclonal, Wuhan, China), anti-pSTAT1 (Tyr701) (1:1000; Cell Signaling Technology, Beverly, MA, USA), anti-STAT1 (1:1000; Protein Tech Group), anti-IRF1 (1:1000; ABclonal), anti-AMPKα (1:1000; Cell Signaling Technology), anti-pAMPKα (Thr172) (1:1000; Cell Signaling Technology) and anti-β-actin (1:1000; Protein Tech Group). After incubation with primary antibodies and washing with TBST, membranes were incubated with IRDye-conjugated secondary antibodies (1:10000; Li-COR Biosciences, Lincoln, NE, USA). Images of immunoreactive bands were captured with Odyssey CLx Infrared Imaging system (Li-COR Biosciences) and quantified with ImageJ software (National Institutes of Health).
6
Western Blot Analysis of Immune Checkpoints
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Tissues or cells were lysed in RIPA buffer (catalog no. KGP702; Keygentec) containing protease and phosphatase inhibitor cocktail (catalog no. P1045; Beyotime) and clarified by centrifugation (12,000×g for 30 min at 4 °C). Protein concentrations were measured using a Pierce BCA Protein Assay Kit (catalog no. KGP902, Keygentec). Protein lysates (30 μg) were subjected to western blotting and protein bands were visualized using a Tianneng imaging system. The following primary antibodies were used: anti-VTCN1 (B7H4) (Proteintech, catalog no. 12080-1-AP), anti-PD-L1/CD274 (Proteintech, catalog no. 66248-1-Ig), anti-IRF1 (Proteintech, catalog no. 11335-1-AP), anti-IFNGR1 (Proteintech, catalog no. 10808-1-AP), anti-IFNGR2 (Proteintech, catalog no. 10266-1-AP), anti-JAK2 (Proteintech, catalog no. 17670-1-AP), anti-STAT1 (Proteintech, catalog no. 10144-2-AP), anti-CD86 (Proteintech, catalog no. 26903-1-AP) and anti-GAPDH (Proteintech, catalog no. 60004-1-Ig). The secondary antibody used was goat anti-mouse IgG (H + L)-HRP (Ray Antibody Biotech, catalog no. RM3001) and goat anti-rabbit IgG (H + L)-HRP (Ray Antibody Biotech, catalog no. RM3002). Because multiple cell lines and target proteins need to be detected, and some proteins have similar molecular weights, the blots have to be cut off before hybridization with antibodies, and Western blotting has to be carried out several times.
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