Cells and brain tissues were extracted, and the protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Proteins were separated using 8 or 12% sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the membrane was blocked with Tris-buffered saline (TBS)/T containing 5% non-fat dry milk and analyzed for the target proteins. The specific antibodies used recognized cleaved-caspase3, p-nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (IκBα) (AP0707, ABclonal, China), p-NFκB (AP0123, ABclonal, China), and p-STAT6 (#56554, CST, United States).
Ap0123
Manufactured by ABclonal
Sourced in China
AP0123 is a laboratory instrument designed for protein purification. It is a chromatography system used to separate and purify proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Ap0707, by ABclonal (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
A2547, by ABclonal (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
A0216, by Beyotime (1 mentions)
A19693, by ABclonal (1 mentions)
A19684, by ABclonal (1 mentions)
A02177 4, by Boster Bio (1 mentions)
A19714, by ABclonal (1 mentions)
Af7022, by Affinity Biosciences (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
A0208, by Beyotime (1 mentions)
Proteome profiler mouse cytokine array, by R&D Systems (1 mentions)
Anti tgfβ sc146, by Santa Cruz Biotechnology (1 mentions)
Ap0475, by ABclonal (1 mentions)
A0980, by ABclonal (1 mentions)
Ac007, by ABclonal (1 mentions)
Vimentin, by Bioss Antibodies (1 mentions)
E ab 20059, by Elabscience (1 mentions)
5 protocols using ap0123
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of Apoptosis and NF-κB Signaling
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Samples were lysed in cell lysis buffer for western and IP (Beyotime). Proteins were quantified by BCA protein assay kit (Beyotime). After complete separation by electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then these membranes were blocked in 5% skimmed milk (Inner Mongolia Yili Industrial Group Co., Ltd., Inner Mongolia, China) for 1 h and incubated with primary antibodies. Then these membranes were incubated with HRP-labeled goat anti-rabbit IgG (H+L) (1: 5,000, A0208, Beyotime) or horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H+L) (1: 5,000, A0216, Beyotime) at room temperature at 37°C for 45 min. Protein bands were visualized with Gel-Pro-Analyzer software (Beijing Liuyi Biotechnology Co., Ltd., Beijing, China).
The primary antibodies used were shown as following: HDAC9 antibody (1: 500, A02177-4, BOSTER, Wuhan, China), cleaved caspase-3 antibody (1:1,000, AF7022, Affinity, Jiangsu, China), Bax antibody (1:1,000, A19684, ABclonal, Wuhan, China), B-cell lymphoma/leukemia-2 (Bcl-2) antibody (1:1,000, A19693, ABclonal), IκBα antibody (1:1,000, A19714, ABclonal), p-IκBα antibody (1:1,000, AP0707, ABclonal), p-p65 antibody (1:1,000, AP0123, ABclonal), p65 antibody (1:1,000, A2547, ABclonal), β-actin (1:1,000, sc-47778, Santa Cruz, Santa Cruz, CA, USA).
The primary antibodies used were shown as following: HDAC9 antibody (1: 500, A02177-4, BOSTER, Wuhan, China), cleaved caspase-3 antibody (1:1,000, AF7022, Affinity, Jiangsu, China), Bax antibody (1:1,000, A19684, ABclonal, Wuhan, China), B-cell lymphoma/leukemia-2 (Bcl-2) antibody (1:1,000, A19693, ABclonal), IκBα antibody (1:1,000, A19714, ABclonal), p-IκBα antibody (1:1,000, AP0707, ABclonal), p-p65 antibody (1:1,000, AP0123, ABclonal), p65 antibody (1:1,000, A2547, ABclonal), β-actin (1:1,000, sc-47778, Santa Cruz, Santa Cruz, CA, USA).
3
Comprehensive Inflammatory Protein Analysis
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Fresh tissues were homogenized and centrifuged and soluble supernatants were taken as whole tissue lysates. Standard Western blots were performed using β-actin as the loading control. Antibodies used were anti-TGF-β (sc-146, Santa Cruz Biotechnology), anti-TNF-α (107,590–1-AP, Proteintech), anti-p65NF-κB (10,745–1-AP, Proteintech), anti-P-p65-S276 (AP0123, Abclonal) and anti-P-p65-S536 (AP0475, Abclonal), all used according to manufacturer recommended dilutions. For Western blot, 10–15 µg of whole lung tissue lysate per sample was used. The relative abundance of a variety of 40 cytokines were examined using mouse cytokine proteome profiler array (ARY028, R&D Systems). The whole tissue lysates of 4 mice in each group were used. Equal amount of total protein 200 µg per sample was hybridized to the array and compared for relative expression. The relative expression was quantified by measuring the dot blot intensity using Image J software.
4
Immunohistochemical Analysis of Bone and Adipose Tissues
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Femora, tibiae, and BAT dissected from the mice were fixed using 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 24 h. Brain dissected from the mice which were transcardially perfused with saline followed by 4% paraformaldehyde (PFA) were post-fixed in 4% PFA at 4°C for 24 h. Femora and tibiae decalcified in 15% EDTA (pH 7.4) at 4°C for 14 days. The tissues were embedded in paraffin, and 5-μm sagittal-oriented of femora, tibiae, and BAT, or coronal-oriented of brain sections were prepared for histological analyses. The sections were incubated with primary antibodies against perilipin 1 (PLIN1, #9349,1:150,CST), P-P65-S276 (AP0123, 1:100, ABclonal, Woburn, MA, USA), Rheb (15924-1-AP, 1:100, Proteintech), or Myd88 (A0980, 1:50, ABclonal) and labeled with secondary antibodies for 1 h in the dark. After labeling, cells were incubated with DAPI for 5 min. Images were acquired with an Olympus 200 M microscope (Olympus, Tokyo, Japan). The numbers of positively-stained cells in the whole medullary space or bone trabecula per femur or tibia in three sequential sections per mouse in each group were counted using Image Pro Plus software.
5
Protein Expression Analysis Protocol
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The cells were lysed with 2% SDS buffer. After transfer, the membrane was incubated with primary antibodies against SLC2A3 (R24453, Zen Bio), E-cad (bs -10,009 R, Bioss), N-cad (bs-1172 R, Bioss), Vimentin (bs-0756 R, Bioss), Snai1 (bs-1371 R, Bioss), and NF-κB P65 (AP0123, ABclonal). GAPDH (E-AB-20059, Elabscience) and α-tubulin (AC007, ABclonal) were used as loading controls. The bands were quantified with ImageJ software and normalized to the loading control.
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