Western blotting was performed according to a standard protocol. Total cell or tissue lysates were extracted using 2% SDS lysis buffer (Applygen, Beijing), and 30 μg of total proteins were separated on 12% (v/v) SDS-PAGE gels. After electrophoresis, the proteins were transferred onto nitrocellulose membranes (Millipore, Ireland) and the membrane was incubated with the anti-CXCL5 antibody (1:500, ab9802, Abcam) and anti-GAPDH antibody (1:1000, H-12; Santa Cruz Biotechnology) at 4 ℃ overnight. After washing three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000, Zsbio, Beijing) for 1 h at room temperature. Finally, the membranes were visualized using an ECL Kit (Applygen, Beijing).
Ab9802
Manufactured by Abcam
Sourced in Ireland, United States, United Kingdom
Ab9802 is a Rabbit Polyclonal Antibody manufactured by Abcam. It is targeted against Human Epidermal Growth Factor Receptor 2 (HER2).
Automatically generated - may contain errors
Lab products found in correlation
Ab86436, by Abcam (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by ZSGB-BIO (1 mentions)
Ecl kit, by Applygen (1 mentions)
Ab136904, by Abcam (1 mentions)
Ab177932, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Ab194699, by Abcam (1 mentions)
Ab56164, by Abcam (1 mentions)
Ab65968, by Abcam (1 mentions)
3 3 diaminobenzidine dab kit, by Agilent Technologies (1 mentions)
Envision detection system, by Agilent Technologies (1 mentions)
4 protocols using ab9802
1
Western Blotting Visualization of CXCL5
2
Evaluating Inflammatory Markers in DSS-Induced Colitis
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To assess the severity of DSS‐induced colitis, the intensity of inflammation and other markers related to fatty acid degradation and cytokines were determined by using IHC analysis. The colon tissues were immune‐stained for anti‐TNF alpha (17590‐1‐ap; Proteintech), anti‐IL‐6 (21865‐1‐AP, Proteintech), anti‐IL‐1β (ab33591, Lianke Biotechnology), anti‐ACOX3 (NBP1‐85901, Novus Biologicals), anti‐ALDH3A2 (15090‐1‐AP, Proteintech), anti‐CXCL5 (ab9802, Abcam), anti‐GRO alpha ( also termed as anti‐CXCL1, ab86436, Abcam), anti‐MIP3a ( also termed as anti‐CCL20, ab136904, Abcam) and anti‐ADH5 (ab177932, Abcam) as previously described.25
3
Western Blotting Quantification Protocol
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The process of western blotting analysis was conducted as described by precious studies [16 (link)]. The protein of sample was extracted by using RIPA Lysis Buffer containing PMSF (Solarbio, China). The protein concentration in each extract was determined using a BCA Protein Quantification Kit (Beyotime, Shanghai, China). Samples of total protein were separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes, which were subsequently blocked with 5% skim milk for 2 h. Subsequently, the membranes were incubated with primary antibodies against CXCL5 (1:1000, ab9802, Abcam, MA, USA) or GAPDH (1:2000, ab181602, Abcam, MA, USA) overnight at 4 °C and then incubated with secondary antibody labeled with HRP (Abcam) for 1 h at 37 °C after washed with TBST for four times. ECL chromogenic substrate was used to visualize the bands, and the Image J software was used to determine the gray value of each band.
4
Quantitative Immunohistochemical Analysis
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Immunohistochemical staining was performed as described [26 (link)]. Formalin-fixed, paraffin-embedded tissue was cut at 4-μm thicknesses and deparaffinized with xylene and ethanol. Endogenous peroxidase activity was blocked by methanol containing 0.3% hydrogen peroxidase. Antigen retrieval was performed by boiling in a microwave oven (citrate buffer, pH 6.0). The sections were incubated overnight at 4°C with primary antibodies against anti-RIP3 (ab56164; Abcam, Cambridge, UK; 1:100), anti-MLKL (ab194699; 1:100), anti-CXCR2 (ab65968; 1:200), anti-CXCL5 (ab9802; 1:500) and anti-CXCL1 (ab86436; 1:100). The immune complexes were then visualized using Envision Detection System (Dako, California, USA) and 3,3′-diaminobenzidine (DAB) Kit (Dako). DAB intensity of MLKL was evaluated as previously described using ImageJ software [27 ]. Images were adjusted by subtracting background as RGB values close to 255 in empty area. Tumor area was selected and mean intensity of DAB color spectrum was calculated, ranging 0 (black) to 255 (total white). The final DAB intensity was calculated according to the formula f = 255—i, where f = final DAB intensity, i = mean DAB intensity. Intensity was calculated in 5 fields at the borders and cores from each patient at 200× magnification under a light microscope.
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