Total proteins were isolated from ESCs and uterine tissues using RIPA buffer (Beyotime, Shanghai, China) [34 (link)]. After quantification with a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and separation by polyacrylamide gel electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen) which were subsequently blocked with 5% defatted milk. After that, the membranes were incubated at 4°C overnight with the following primary antibodies: anti-SOCS3 (ab280884, 1:1000), anti-β-actin (ab115777, 1:200), anti-p-JAK2 (ab32101, 1:1000), anti-JAK2 (ab108596, 1:5000), anti-p-STAT3 (ab267373, 1:1000), anti-STAT3 (ab68153, 1:1000) (all from Abcam) followed by being incubated with the horseradish peroxidase-conjugated secondary antibody of goat anti-rabbit IgG H&L (Abcam, ab6702, 1:1000) at room temperature for 2 h. The proteins were visualized using an ECL kit (Cwbiotech, Beijing, China) and quantified with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).
Ab280884
Manufactured by Abcam
Sourced in China, United States, United Kingdom
Ab280884 is a reagent used for laboratory research. It is a primary antibody that specifically recognizes and binds to a target antigen. The core function of this product is to facilitate the detection and study of the target antigen in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab68153, by Abcam (3 mentions)
Ab108596, by Abcam (3 mentions)
Ab32101, by Abcam (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab115777, by Abcam (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ecl kit, by CWBIO (1 mentions)
Ab267373, by Abcam (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Horseradish peroxidase conjugated secondary antibody of goat anti rabbit igg h l, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Beyotime (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab76302, by Abcam (1 mentions)
Ab195049, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab32536, by Abcam (1 mentions)
Ab76315, by Abcam (1 mentions)
6 protocols using ab280884
1
Protein Isolation and Western Blot Analysis
2
Lung Tissue Protein Analysis
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Lung tissues (50 mg per mouse) were homogenized in ice‐cold RIPA buffer (Sigma‐Aldrich) with a 1% protease inhibitor cocktail and a phosphatase inhibitor, and protein concentration was examined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Thereafter, proteins (40 μg) were separated by 10% SDS‐PAGE and blotted onto a PVDF membrane. After blocking with 5% skimmed milk for 2 h, the membrane was incubated overnight with primary antibodies against p‐JAK2 (ab32101, 1:4000; Abcam), JAK2 (ab108596, 1:5000; Abcam), phosphorylated STAT3 (ab76315, 1:5000; Abcam), STAT3 (ab68153, 1:2000; Abcam), SOCS3 (ab280884, 1:1000; Abcam), p‐NF‐κB p65 (ab76302, 1:1000; Abcam), NF‐κB p65 (ab32536, 1:1000; Abcam), p‐p38 MAPK (ab195049, 1:1000; Abcam), p38 MAPK (ab170099, 1:1000; Abcam), and GAPDH (ab9485, 1:2500; Abcam) at 4°C, and subsequently incubated with secondary antibodies for 2 h at room temperature. The bands were visualized by enhanced chemiluminescence reagent (Beyotime), and the intensity of blot was quantified by Image Lab 3.0 software (Bio‐Rad, Hercules, CA, USA).
3
SOCS3 Protein Detection in BMSCs
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RIPA buffer (Beyotime, Shanghai, China) was used to extract total protein in BMSCs. The protein concentration was assessed by a BCA Protein Assay Kit. Then the collected proteins (40 μg) were isolated with electrophoresis on 10% SDS-PAGE and transferred onto PVDF membranes. Subsequently, the membranes were blocked with 5% non-fat milk and then cultured with primary antibody of anti-SOCS3 (#ab280884, 1/1000, Abcam) overnight at 4 °C. Next, the membranes were further incubated with the secondary antibodies in the dark for 2 h at room temperature. The protein bands were measured by a chemiluminescence detection system (Amersham, Little Chalfont, UK).
4
SOCS3 Modulation of TLR4 Expression
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HK-2 cells were transfected with pCMV empty vector or pCMV SOCS-3 for 48 h. Then, cells were lysed using IP Lysis/Wash Buffer supplemented with Halt™ Protease (Product No. 78, 430) and Phosphatase (Product No. 78,428) Inhibitor Cocktails. Subsequently, Co-Immunoprecipitation was performed using a commercial Pierce™ Co-Immunoprecipitation Kit (PI26149, Thermo Scien-tific™, MA, USA) according to the detailed manual. The rabbit anti-SOCS3 (ab280884, Abcam, MA, USA) and rabbit anti-TLR4 (19811-1-AP, Proteintech, CA, USA) were used individually to detect the expressions of SOCS3 and TLR4.
5
Western Blot Analysis of Renal Proteins
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Rat renal tissues were homogenized in 0.5 mL of radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio) for protein separation. The protein concentration of each sample was estimated using a BCA protein assay kit (Vazyme, Nanjing, China). For detection of protein levels, protein lysates (40 μg protein loaded per lane) for each group of tissues were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene fluoride (PVDF) membranes followed by blocking using 5% nonfat milk. After 1 h of blocking, the membrane was incubated overnight with primary antibodies against IGF-1R (ab182408, 1:1,000; Abcam), β-actin (ab115777, 1:200; Abcam), COX2 (ab179800, 1:2,500; Abcam), iNOS (ab178945, 1:1,000; Abcam), TGF-β (ab179695, 1:1,000; Abcam), collagen IV (ab6586, 1:500; Abcam), fibronectin (ab45688, 1:3,000; Abcam), p-JAK2 (ab32101, 1:2,000; Abcam), JAK2 (ab108596, 1:5,000; Abcam), p-STAT1 (ab109461, 1:5,000; Abcam), STAT1 (ab230428, 1:750; Abcam), p-STAT3 (ab32143, 1:5,000; Abcam), STAT3 (ab68153, 1:2,000; Abcam), SOCS1 (ab65989, 1 μg/mL; Abcam), and SOCS3 (ab280884, 1:1,000; Abcam) at 4°C. Then, the membrane was incubated with anti-rabbit (ab97051, 1:20,000; Abcam) at room temperature for 2 h. The proteins were visualized with an enhanced chemiluminescence detection kit (Beyotime, Shanghai, China).
6
Macrophage and Angiogenesis Profiling
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Tissue samples were embedded in paraffin. H&E and Masson's trichrome were used to stain sections. After the slides were incubated with 5 % BSA solution for 60 min, CD68 (universal macrophage maker) antibody solution (1:200, ab283654 ; Abcam, UK) and CD163 (M2 phenotype of macrophage marker) antibody solution (1:300, ab182422; Abcam, UK) were incubated with the sections. CD31 staining (1:300, ab182981; Abcam, UK) was used to evaluate angiogenesis changes. SOCS3 staining (1:500, ab280884; Abcam, UK) was used to evaluate expression changes.
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