Protein concentrations from cultured ESCs were quantified using the BCA protein assay kit (P0010S, Beyotime, China). Equal amount of protein (30μg) was subjected to 12% sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (0.45mm, Millipore, USA). After blocked for 1 hour at room temperature (RT) with blocking buffer (0.1% Tris-buffered saline with Tween (TBST) with 5% fat-free dried milk powder), the blots were incubated with primary antibodies against GPER (1: 500, ab39742, Abcam) or HIF-1α (1:1000, #AF1009, Affinity) at 4°C for overnight. The target proteins were visualized by the ECL western blotting detection system (Millipore) after incubation with a secondary antibody (1:5000 diluted with 5% fat-free dried milk powder in 0.1% 5 TBST).
Af1009
Manufactured by Affinity Biosciences
Sourced in United States, China
AF1009 is a laboratory equipment product from Affinity Biosciences. It is designed for the purpose of performing affinity-based analytical procedures. The core function of AF1009 is to facilitate the separation, purification, and analysis of biomolecules such as proteins, peptides, and other macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Ab39742, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Af5186, by Affinity Biosciences (2 mentions)
Af6308, by Affinity Biosciences (2 mentions)
Af3308, by Affinity Biosciences (2 mentions)
Ap53886pu n, by OriGene (2 mentions)
A0216, by Beyotime (2 mentions)
A0208, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ecl western blotting detection system, by Merck Group (1 mentions)
Af5166, by Affinity Biosciences (1 mentions)
Af7001, by Affinity Biosciences (1 mentions)
Bs 1313r, by Bioss Antibodies (1 mentions)
Df7731, by Affinity Biosciences (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Molecular weight marker, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Af5131, by Affinity Biosciences (1 mentions)
13 protocols using af1009
1
Quantifying ESC Protein Levels
2
Western Blot Analysis of Callus Proteins
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The proteins from the callus tissues were extracted by using lysis buffer (Beyotime, Shanghai, China). Then the protein was quantified by the BCA assay kit (Beyotime, Shanghai, China). After that, an equal amount of proteins was electrophoresed on SDS-PAGE and then transferred onto PVDF membranes (Thermo Fisher, Cambridge, MA, USA). Next, the membranes were immuno-blotted with Hypoxia-inducible factor-1α (HIF-1α) rabbit antibody (1: 500 dilution, Catalog No.: AF1009, Affinity, Changzhou, Jiangsu, China), vascular endothelial growth factor (VEGF) rabbit antibody (1: 500 dilution, Catalog No.: bs-1313R, Bioss, Beijing, China), Runx2 rabbit antibody (1: 500 dilution, Catalog No.: AF5186, Affinity), Osterix (1: 400 dilution, Catalog No.: DF7731, Affinity), Type I collagen antibody (1: 1000 dilution, Catalog No.: AF7001, Affinity), SDF-1α rabbit antibody (1: 500 dilution, Catalog No.: AF5166, Affinity), CXCR4 antibody (1: 1000 dilution, Catalog No.: A12534, ABclonal, Wuhan, China), or β-actin mouse antibody (1: 2000 dilution, Catalog No.:60008-1-Ig, proteintech, Wuhan, Hubei, China). The membranes were washed three times and incubated with goat HRP-conjugated secondary antibodies (proteintech, Wuhan, Hubei, China). The specific bands were developed with the ECL system (7 Sea biotech, Shanghai, China). β-actin was used as the control of HIF-1α and VEGF.
3
Western Blot Analysis of Gastric Tissue
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Total protein was extracted from the gastric tissue and SNU‐5 and AGS cell lines using conventional methods. Equal amounts of protein samples (15 μg each) and molecular weight markers (Thermo Fisher, USA) were separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and electrically transferred to a poly (vinylidene fluoride) membrane, which was then blocked in 5% skim milk for 2 h and incubated with the primary antibody overnight at 4°C. After incubation with secondary antibodies for 2 h at 25°C, protein bands were visualized using an enhanced chemiluminescence detection kit (Thermo Fisher, USA). They were also washed appropriately between each step using Tris‐buffered saline containing 0.1% Tween‐20 (TRIS‐buffered saline containing 0.1% Tween‐20). The following antibodies were used: KIRREL (BS‐6435R, Bioss, China), PI3K (AF6241, Affinity, China), P‐PI3K (AF3242, Affinity, China), AKT (AF6261, Affinity, China), P‐AKT (AF0832, Affinity, China), m‐TOR (AF6308, Affinity, China), P‐mTOR (AF3308, Affinity, China), HIF‐1α (AF1009, Affinity, China), VEGF (AF5131, Affinity, China) and GAPDH (TA‐08, ZSGB‐BIO, China).
4
Immunofluorescence Analysis of Lung AT2 Cells
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Lung tissue was fixed with 4% PFA for at least 48 h, and then dehydrated and embedded in paraffin. The embedded lung tissue was sectioned into slices with a thickness of 4 μm and subsequently affixed onto slides for staining. AT2 cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then permeabilized with 0.5% Tryon-100 for 30 min. Cells washed with PBS were blocked with 5% bovine serum albumin (BSA) (SW3015, Solarbio, Beijing, China) for 1 h at room temperature.
Immunofluorescence staining was performed using primary antibodies against SPC (1:200, AP53886PU-N, OriGene, Maryland, MD, USA), Ki67 (1:200, GTX00538, Genetex, Texas, USA), or HIF-1α (1:100, AF1009, Affinity, Changzhou, China). Following overnight incubation at 4 °C, the slides were washed and subsequently incubated with the corresponding secondary antibody, IgG Alexa Fluor 594 (1:1000, 8889 s, Cell Signaling Technology, Boston, MA, USA), for 1 h at 37 °C in a light-restricted environment. This was followed by a 5 min incubation with 4′,6-diamidino-2-phenylindole (DAPI, 10 μg/mL) in darkness. Images were promptly captured using a fluorescence microscope.
Immunofluorescence staining was performed using primary antibodies against SPC (1:200, AP53886PU-N, OriGene, Maryland, MD, USA), Ki67 (1:200, GTX00538, Genetex, Texas, USA), or HIF-1α (1:100, AF1009, Affinity, Changzhou, China). Following overnight incubation at 4 °C, the slides were washed and subsequently incubated with the corresponding secondary antibody, IgG Alexa Fluor 594 (1:1000, 8889 s, Cell Signaling Technology, Boston, MA, USA), for 1 h at 37 °C in a light-restricted environment. This was followed by a 5 min incubation with 4′,6-diamidino-2-phenylindole (DAPI, 10 μg/mL) in darkness. Images were promptly captured using a fluorescence microscope.
5
Quantification and Profiling of Protein Markers
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The total proteins were extracted using a RIPA assay kit. Total protein was quantified by BCA Protein Assay Kit (Beyotime, China). Subsequently, the proteins were separated via SDS-PAGE and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Pall, USA). The membranes were blocked with the QuickBlock™ Blocking Buffer (P0252; Beyotime Biotechnology) at room temperature for 15 min at room temperature, and blots were then incubated at 4 °C with the following diluted primary rabbit anti-human antibodies: anti-IGF1R (1:3000, K106546P; Solarbio), anti-EGR1 (1:600, D120585; BBI), anti-OBCAL (1:1000, DF8584; Affinity), anti-TGF beta1 (1:1000, AF1027; Affinity), anti-TGF beta2 Ab (1:1000, AF0260; Affinity), anti-Smad2/3 (1:1000, AF6367; Affinity), anti-Smad4 (1:1000, AF5247; Affinity), anti-HIF1A (1:1000, AF1009; Affinity), and anti-beta Actin (1:5000, AF7018; Affinity), followed by horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) H&L (1:5000; ZB-2301; ZSG-BIO) for 2 h at room temperature. After washing three times in TBST, bands were visualized utilizing BeyoECL Plus Moon (P0018S; Beyotime Biotechnology). The immunoblotting results were analyzed using the Image J software.
6
Immunohistochemical Evaluation of GPER and HIF-1α
7
Protein Expression Analysis in Cells
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By adding 1 mL of RIPA buffer and 10 µL of benzylmethylsulfonyl fluoride (Solarbio, Beijing, China), total protein was extracted. The quantification of all proteins was performed using an enhanced BCA protein assay kit (Thermo Fisher Scientific, Niederelbert, Germany). Following denaturation, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred to a polyvinylidene fluoride membrane and blocked with 5% skim milk at 24 °C for 2 h. The membrane was then incubated with polyclonal antibodies against SPC (1:1000; AP53886PU-N; OriGene, Maryland, USA), HIF-1α (1:1000; AF1009; Affinity, Changzhou, China), EGFR (1:300; bs-10007R; Bioss, Beijing, China), EGF (1:300; bs2010R; Bioss Beijing China), PCNA (1:300; bs-2006R; Bioss Beijing China), Bax (1:300; bs-20386R; Bioss Beijing China), and β-actin (1:2000, Bioss, Beijing, China). After washing with PBS at 24 °C for 90 min, the membrane was further incubated with IgG antibody (1:3000, bs-0295G-HRP, Bioss, Beijing, China). Finally, an ECL luminescent solution was used for color development and the results were observed. β-actin served as the loading control. Grayscale values of protein bands were analyzed using ImageJ software to determine relative expression levels.
8
Protein Expression Analysis in NSCLC Cells
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Briefly, PMSF and RIPA lysis buffer (Beyotime) were employed to extract the NSCLC cell proteins. To detect the concentration of cell proteins, Pierce BCA Protein assay kit was conducted. Cell proteins were isolated by 10% SDS‐PAGE before transferring to PVDF membranes. Using 5% skim milk, we blocked PVDF membranes at room temperature for 1 h. Later, with primary antibodies, the membranes were incubated overnight at 4°C, including UCP2 (1:1000, #DF8626, Affinity, USA), mTOR (1:500, #AF6308, Affinity), p‐mTOR(Ser2448) (1:500, #AF3308, Affinity), S6K (1:500, #AF6226, Affinity), p‐S6K(Thr389) (1:500, #AF3228, Affinity), 4E‐BP (1:500, #AF6432, Affinity), p‐4E‐BP(Thr70) (1:500, #AF2308, Affinity), HIF‐1α (1:500, #AF1009, Affinity) and α‐Tubulin (1:1000, #AF4651, Affinity). Next day, with HRP‐linked secondary antibody (1:3000, #S0001, Affinity), the membranes were washed before incubation at room temperature for 2 h. Finally, the membranes were visualized by ECL Detection Reagent (Yeasen, China), and ImageJ software was used to quantify the relative grayscale value.
9
Western Blot Analysis of Protein Markers
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Cells were lysed by PMSF added lysate (ST506 and P0013, Beyotime, China). Protein concentration was measured by a BCA detection kit (P0011, Beyotime, China). Then, the protein was subjected to SDS-polyacrylamide gel electrophoresis (P0015, Beyotime, China) and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore, USA). After blocked with 5% nonfat milk (YiLi, China) for 1 h at room temperature, the membrane was incubated with primary antibody overnight at 4 °C, followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit (A0208, Beyotime, China) or anti-mouse (A0216, Beyotime, China) antibody at 1:5000 dilution for 45 min. The blots were incubated in ECL substrate (P0018, Beyotime, China), and images were analyzed using the Gel-Pro-Analyzer software.
Antibody information: NREP (1:500, PA5-68426, Thermofisher, USA), HIF-1α (1:500, AF1009, Affinity, China), E-cadherin (1:1000, A20798, Abclonal, China), N-cadherin (1:500, A19083, Abclonal, China), SLUG (1:1000, A1057, Abclonal, China), MMP9 (1:500, A0289, Abclonal, China), PKM2 (1:500, A13905, Abclonal, China), GLUT1 (1:1000, AF5462, Affinity, China), HK-2 (1:500, A20829, Abclonal, China), and LDHA (1:500, A1146, Abclonal, China).
Antibody information: NREP (1:500, PA5-68426, Thermofisher, USA), HIF-1α (1:500, AF1009, Affinity, China), E-cadherin (1:1000, A20798, Abclonal, China), N-cadherin (1:500, A19083, Abclonal, China), SLUG (1:1000, A1057, Abclonal, China), MMP9 (1:500, A0289, Abclonal, China), PKM2 (1:500, A13905, Abclonal, China), GLUT1 (1:1000, AF5462, Affinity, China), HK-2 (1:500, A20829, Abclonal, China), and LDHA (1:500, A1146, Abclonal, China).
10
Immunohistochemical Analysis of Murine Knee Cartilage
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Knee joint sections of mice were deparaffinized and moved in 2 changes of xylene each for 10 min, hydrated in a series of ethanol (100% ethanol, 95% ethanol, followed by one shift of 70% ethanol), and rinsed twice in PBS. The antigen was retrieved by retrieval kit (AR0022; Boster) for the sections (30min; 37 °C). H2O2 (0.5%) was used to quench endogenous peroxidases for 10 min. The tissue sections were incubated with selected primary antibodies (overnight; 4 °C). Slides were rinsed in PBS containing 0.1% tween 20 (PBST, catalog P1397, Millipore Sigma) and antigen blocked with 5% normal goat serum. Next, overnight incubation at 4 °C with rabbit anti-mouse ACAN (1:200 dilution, catalog DF7561, Affinity), mouse anti-mouse Runx2 (1:200 dilution, Affinity; AF5186), rabbit anti-mouse MMP-13 (1:200 dilution, catalog 18165-1-AP, Proteintech), HIF-1α (1:200 dilution, Affinity; AF1009) and ATF4 (1:200 dilution, Affinity; DF6008). Slides were incubated with anti-rabbit secondary antibody for 30 min at room temperature, 3 times PBS rinsed for 5 min each, followed by two washes in deionized water for 5 min each. A 3-min incubation detected antibody binding to ACAN, MMP13, Runx2, HIF-1α, and ATF4 antigen with DAB peroxidase substrate (catalog zli90181, OriGene). Mayer's hematoxylin solution (catalog AR0005, BOSTER) was used to counterstain nuclei for 10–15 s.
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