For the extraction of total protein, 100 μL RIPA lysis buffer (CW2334S; CWBIO, Beijing, China) and 1 μL Protease Inhibitor Cocktail (CW2200S; CWBIO, Beijing, China) were added to 100 mg of jejunal tissue, and the total protein was quantified using a bicinchoninic acid protein assay kit (CW0014; CWBIO, Beijing, China). Protein was separated by SDS-PAGE (Solarbio, Beijing, China) (20 μg per lane) and then transferred onto PVDF membranes (IPVH00010; Millipore, Danvers, MA). Next, the membranes were incubated with polyclonal goat anti-Wnt3 (ab116222; Abcam, UK), polyclonal rabbit anti-β-catenin (ab6302; Abcam, UK), monoclonal rabbit anti-TCF4/TCF7L2 (2565; CST, Boston, MA), polyclonal rabbit anti-ZO-1 (61-7300; Invitrogen, Camarillo, CA), and polyclonal rabbit anti-Claudin (ab129119; Abcam, UK) (all 1:2000 in Tris-buffered saline with Tween) overnight at 4°C. The membranes were incubated with HRP-conjugated rabbit anti-goat IgG (H&L) (1:5000; bs-0294R; Beijing Bioss Biotechnology Co., Ltd., Beijing, China) or HRP-conjugated goat anti-rabbit IgG (H&L) (1:5000; K008; Kmbio, Beijing, China) for 1 h at 37°C. Polyclonal anti-β-actin (1:5000; ab119716; Abcam, UK) was used for normalization of band intensities. The densitometric values of obtained immunoblot signals from 6 separate experiments were determined using Image J (National Institutes of Health, New York).
Ab129119
Manufactured by Abcam
Sourced in United States
Ab129119 is a product offered by Abcam. It is a laboratory equipment item, but a detailed description of its core function is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab119716, by Abcam (1 mentions)
Ab6302, by Abcam (1 mentions)
Ab116222, by Abcam (1 mentions)
Sds page, by Solarbio (1 mentions)
Cw2334s, by CWBIO (1 mentions)
Cw2200, by CWBIO (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Cw0014, by CWBIO (1 mentions)
Anti flag 20543 1 ap, by Proteintech (1 mentions)
Anti occludin 13409 1 ap, by Proteintech (1 mentions)
Bs 0295g hrp, by Bioss Antibodies (1 mentions)
Anti zo 1 21 773 1 ap, by Proteintech (1 mentions)
Ab199529, by Abcam (1 mentions)
Bs 0296g hrp, by Bioss Antibodies (1 mentions)
Anti gapdh 10494 1 ap, by Proteintech (1 mentions)
A0516, by Beyotime (1 mentions)
Ab167161, by Abcam (1 mentions)
Ab15104, by Abcam (1 mentions)
Ab96587, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
3 protocols using ab129119
1
Analyzing Intestinal Wnt Signaling Pathway
2
Antibody Validation for Western Blot and Immunofluorescence
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For Western blot or immunofluorescence analysis, the following antibodies were used: anti-ADAM10 (A10438; ABclonal, Wuhan, China), anti-E-cadherin full length (3195; Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin NTF (A3044, ABclonal), anti-claudin-1 (ab129119; Abcam, Cambridge, MA, USA), anti-occludin (13,409-1-AP; Proteintech, Wuhan, China), anti-ZO-1 (21,773-1-AP; Proteintech), anti-HtrA1 (ab199529; Abcam), anti-MMP7 (10,374-2-AP; Proteintech), anti-FLAG (20,543-1-AP; Proteintech), and anti-GAPDH (10,494-1-AP; Proteintech). Horseradish peroxidase (HRP)-labeled goat anti-rabbit (bs-0295 G-HRP; Bioss, Beijing, China), HRP-labeled goat anti-mouse (bs-0296 G-HRP; Bioss), and Cy3-conjugated goat anti-rabbit (A0516; Beyotime) secondary antibodies were used.
3
Western Blot Analysis of Tight Junction Proteins
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The western blotting was performed as Linshi reported methods and modification. RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) and extraction kits (ThermoFisher Scientific, Shanghai, China) were used to isolate the total proteins, a nuclear protein, and cytoplasmic proteins, respectively. The protein concentration was analyzed using a BCA reagent (CWBIO, Beijing, China) followed by SDS-PAGE and electrotransfer of separated protein lysates to nitrocellulose membranes (Millipore). After 1 h of blocking, the membranes were incubated with a primary antibody at 4°C for overnight, then incubated with the secondary antibody for 2 h at room temperature. Positive bands were measured by enhanced chemo-luminescence (Tanon, Shanghai, China). Gel-Pro Analyzer software version 4.0 (Media Cybernetics, Silver Spring, MD, USA) was used to analyse the densitometry. Antibodies against ZO-1 (ab96587), occludin (ab167161), claudin-1 (ab129119), and claudin-4 (ab15104) were obtained from Abcam (Cambridge, MA, USA). The antibody against ERK (4695S), P-ERK (4370S), and β-actin (4970S) were secured from CST (CST, MA, USA).
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