The total proteins in ileum tissues were extracted by Radio-Immunoprecipitation Assay (RIPA, 89901, Thermo Scientific, USA). The protein concentration was detected by the BCA method, and the proteins (25 μg in each lane) were electrophoresed in 6% polyacrylamide gel. The isolated proteins were then transferred onto a PVDF membrane. The membrane was blocked for 1 h at room temperature for nonspecific binding with 5% bovine serum albumin (BSA). After that, the membrane was incubated with rabbit anti-zonula occludens- (ZO-) 1 antibody (1 : 1000, ab96587, Abcam, UK), rabbit anti-Claudin-1 antibody (1 : 1000, ab15098, Abcam, UK), anti-NF-κB p65 (1 : 5000, ab207297, Abcam, UK), and rabbit anti-GAPDH antibody (1 : 1000, ab181602, Abcam, UK) at 4°C for the night. The membrane was exposed into Goat Anti-Rabbit IgG H&L (1 : 5000, ab207297, Abcam, UK) at room temperature for 1 h, and blots were performed using ECL detection reagents (Merck Millipore, USA).
Ab207297
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab207297 is an antibody product from Abcam. It is a purified rabbit monoclonal antibody.
Automatically generated - may contain errors
Lab products found in correlation
Ab239882, by Abcam (3 mentions)
Ab8226, by Abcam (3 mentions)
Ab15191, by Abcam (2 mentions)
Ab9056, by Abcam (2 mentions)
Ab15323, by Abcam (2 mentions)
Ab205719, by Abcam (2 mentions)
Ab32518, by Abcam (2 mentions)
Ab13847, by Abcam (2 mentions)
Ab254360, by Abcam (2 mentions)
Ab133739, by Abcam (2 mentions)
Ab13556, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab179800, by Abcam (2 mentions)
Ab222494, by Abcam (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Protein phosphatase inhibitor, by Merck Group (1 mentions)
Las 4000 imaging analyzer, by Fujifilm (1 mentions)
Ab179461, by Abcam (1 mentions)
Ab45381, by Abcam (1 mentions)
18 protocols using ab207297
1
Intestinal Tight Junction Protein Analysis
2
Inflammatory Protein Expression in Hippocampus
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Hippocampal tissues were isolated from mice of each group (n = 10) and then lysed with RIPA buffer containing protein phosphatase inhibitor (Sigma-Aldrich). Total proteins were extracted and transferred onto a PVDF membrane, followed by blockage for 2 h, and incubation with primary antibodies [IL-17A antibody (ab9056), iNOS antibody (ab15323), NF-κB p65 antibody (ab207297), and COX-2 antibody (ab15191)(all 1:1,000; Abcam) and secondary antibodies. In this study, β-actin served as the internal reference protein. The ultra-sensitive chemiluminescent liquid-based FujiFilm LAS 4000 imaging analyzer (FujiFilm, Tokyo, Japan) was used for visualization, and Image J (NIH, Bethesda, MD) was used to analyze the relative intensities of individual bands.
3
Salivary Gland Protein Expression Analysis
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Total proteins were extracted from salivary glands tissues using RIPA lysis buffer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%) was used for protein electrophoresis. The proteins were then transferred to the polyvinylidene difluoride (PVDF) membranes. After blocking with 5% bovine serum albumin, the membranes were incubated with diluted primary antibodies for 12 h at 4°C. After washing, the PDVF membranes were incubated with diluted goat anti-rabbit IgG H&L (HRP) (1:5,000, ab6721; Abcam), goat anti-mouse IgG H&L (HRP) (1:5,000, ab205719; Abcam) at room temperature for 2 h. The bands were detected using the ECL system (Bio-Rad) and analyzed using Image J. The primary antibodies used were p-NF-κB (1:1,000, ab239882; Abcam), NF-κB (1:1,000, ab207297), p-ERK (1:1,000, ab201015), ERK (1:10,000, ab184699), p-JNK (1:5,000, ab76572), JNK (1:1,000, ab179461), p-P38 (1:1,000, ab45381), P38 (1:1,000, ab170099), and β-actin (1 µg/ml; ab8226).
4
Western Blot Analysis of Osteoblast Markers
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The total proteins of MC3T3-E1 cells or BMSCs were extracted and lysed with RIPA buffer (Beyotime, China), and protein concentration was detected using a BCA protein assay kit (Solarbio, China). Then, a 20 μg total protein sample was loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation and transferred to a PVDF membrane. After being blocked with 5% skimmed milk, the membrane was incubated at 37°C with the primary antibodies against caspase-3 (ab13847, Abcam), caspase-9 (ab32539, Abcam), SIRT1 (ab110304, Abcam), BMP-2 (ab214821, Abcam), RANKL (ab45039, Abcam), OPG (ab73400, Abcam), IκBα (ab32518, Abcam), p-IκBα (AF2002, Affinity), p65 (ab207297, Abcam), p-p65 (AF2006, Affinity), IKKα (ab32041, Abcam), p-IKKα (AF3013, Affinity), NFATc1 (ab2796, Abcam), and cathepsin K (ab19027, Abcam) at 4°C overnight. After that, the membrane was further incubated with the secondary antibody at room temperature for 1 h. Then, the enhanced chemiluminescence (ECL) reagent (Solarbio, China) was used to visualize the protein bands, and the relative band density was semiquantified with the ImageJ software.
5
Western Blot Analysis of Cellular Proteins
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Total protein samples were extracted from cell samples with RIPA buffer (CWBio, Beijing, China) and protein concentration was measured using BCA protein assay kit (Beyotime, Shanghai, China). Equal amount of protein sample was separated on 12% SDS-PAGE gels followed by transferred to nitrocellulose membranes. Then, the membranes were blocked in 5% non-fat milk dissolved in TBST solution and incubated with primary antibodies against p300 (Abcam, Cambridge, MA, USA; ab275378, 1:1000 diluted), RhoA (ab187027, 1:2000 diluted), ROCK (ab134181, 1:1000 diluted), NF-κB p65(ab207297, 1:1000 diluted), HDAC1 (ab109411, 1:4000 diluted), COL1A1 (ab270993, 1:3000 diluted), COL2A1 (ab34712, 1:3000 diluted), and GAPDH (ab8245, 1:5000 diluted) overnight at 4°C. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 2 h. The protein bands were visualized using an enhanced chemiluminescence reagent (Pierce Biotech, Inc., Rockford, IL, USA).
6
Western Blot Analysis of Inflammatory Signaling
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After lysing samples with RIPA lysis buffer (SY4680, YITA), the concentration of protein was determined using a Dxy_PIERCE BCA Protein Assay kit (23227, Thermo Scientific). Equal amounts of protein specimens (50 µg/g/lane) were resolved on 12.5% polyacrylamide gels at 130 V and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 2.5% non-fat milk at 37°C for 1 h and then probed with primary antibodies against TLR4 (HTA125, 1 : 1000, eBioscience, CA, USA), NF-κB p65 (ab207297, 1 : 1000, Abcam), p-NF-κB p65 (ab76302, 1 : 1000, Abcam), IL-1β (ab254360, 1 : 1000, Abcam), and GAPDH (ab181603, 1 : 10000, Abcam) in a 4°C refrigerator overnight with slow shaking. After rinsing with TBST three times, the membranes were incubated with secondary antibodies (ab288151, 1 : 10000, Abcam) at 37°C for 1 h. After washing the membranes again, the proteins were visualized using the ECL detection system (JMCDVISSP, Millipore) in accordance with the product manuals and quantified with ImageJ software v.1.52v (National Institutes of Health).
7
Western Blot Analysis of Apoptosis Markers
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The cell or tissue sample was lysed with an RIPA lysis buffer containing 1 mmol/L PMSF. Then, the homogenates were prepared and centrifuged at 12,000 rpm for 10 min at 4°C. The protein concentration was determined by the BCA method. Non-specific binding was blocked with 5% skimmed milk for 1.5 h, and the membranes were incubated with diluted primary antibodies against Caspase-3 (ab32042, 1:800; Abcam, Cambridge, MA, United States), TLR4 (ab13556, 1:800; Abcam), MyD88 (ab133739,1:800; Abcam), NF-κB p65 (ab207297, 1:800; Abcam), and β-actin (ab8226, 1:1,000; Abcam) overnight at 4°C and in the secondary antibody for 1 h at room temperature. The signals were determined by the Amersham prime ECL Plus detection system (Pittsburgh, PA).
8
Quantification of NF-κB Signaling
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The antibodies for phospho nuclear factor kappa B (p65-NF-κB ) (E379, ab207297), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (p-IκBα ) (6A920, ab12134) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH ) (EPR16891, ab181602) were purchased from Abcam, Cambridge, Massachusetts, USA. The protein expressions of p65-NF-κB and p-IκBα were estimated in spinal cord tissue according to the method described elsewhere [39 ].
9
Western Blot Analysis of Inflammasome Pathway
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As described in our previous study [24 (link)], the whole cell lysate was harvested and subjected to SDS-PAGE and transferred into the nitrocellulose transfer membrane. After incubation with 5% (w/v) milk in PBS/0.05% (v/v) Tween-20 for 1 hour, the membrane was incubated with indicated antibodies overnight at 4°C, subsequently followed by incubation with a horseradish peroxidase secondary antibody (Jackson ImmunoResearch) for 1 hour at room temperature. Proteins were detected using an enhanced chemiluminescence (PerkinElmer). The antibodies used in this study were from Abcam: NLRP3 (ab263899, 1 : 2000 for WB), IL-1β (ab254360, 1 : 1000 for WB), caspase-1 (ab179515, 1 : 1000 for WB), ASC (AB283684,1 : 2000 for WB), GSDMD (ab219800, 1 : 2000 for WB), NF-κB (ab207297, 1 : 2000 for WB), phospho-NF-κB (ab239882, 1 : 2000 for WB), β-actin (ab8226, 1 : 4000 for WB), and α-tubulin (ab7291, 1 : 5000 for WB).
10
Immunohistochemistry of NF-κB p65
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Briefly, paraffin sections were dewaxed and incubated with 3% hydrogen peroxide (H2O2) to block the endogenous peroxidase activity. Pepsin K was applied following the exposure to anti-NF-κB p65 antibody (1 : 1000, ab207297, Abcam, UK) at 4°C overnight. After incubation with the HRP-conjugated secondary antibody (Goat Anti-Rabbit IgG H&L, ab205718, Abcam, UK), DAB (ab64238, Abcam, UK) was used for immunohistochemical staining. The slides were then dehydrated in graded alcohol and sealed. The images were acquired under the light microscopy (TS100, Nikon, Japan).
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