The colonic tissues of 5 rats were randomly selected from each group and subjected to histopathological analysis after the fixing of the colon specimen in 10% formalin in PBS followed by embedding in paraffin. Approximately 4 mm-thick sections of the colon were prepared, stained with Eosin and Hematoxylin, and then observed under a light microscope. All sections were analyzed and interpreted by a certified histopathologist. The remaining colonic tissues were homogenized in a commercial Pro-Prep Protein Extraction Solution (Shanghai Beyotime, China). The relative parameters were determined using Elisa kits according to the manufacturer's instructions.
Pro prep protein extraction solution
Manufactured by Beyotime
Sourced in China
Pro-Prep Protein Extraction Solution is a laboratory reagent designed for the efficient extraction of proteins from biological samples. It is a buffered solution containing proprietary components that facilitate the solubilization and recovery of proteins from various sources, such as cells, tissues, or organisms. The core function of this product is to provide a reliable and standardized method for extracting proteins for further analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Nf κb, by Abcam (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
P gsk3β ser9, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Ecl detection system, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Image lab version 3.1, by Bio-Rad (1 mentions)
Pierce ecl western blot substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl detection reagent, by Thermo Fisher Scientific (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
5 protocols using pro prep protein extraction solution
1
Histopathological Analysis of Rat Colonic Tissues
2
Protein Extraction and Western Blot Analysis
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Protein samples were prepared using PRO‐PREP protein extraction solution (Beyotime Institute of Biotechnology) for total fractions according to the manufacturer's instructions.12 Total protein was separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% non‐fat milk for 1 hour, incubated overnight at 4°C with antiserum against CARD9, NF‐κBp65, p38MAPK, TLR4, Dectin‐1 (1:1000 respectively, Abcam), p‐NF‐κBp65, p‐p38MAPK (1:1000 respectively, CST), and then incubated with the secondary antibodies for 1 hour. Antibodies against β‐actin (1:10 000, Abcam, Cambridge, UK) or GAPDH (1:5000, Abcam) were used as an internal control. Then, chemiluminescence detection was carried out, using ECL Plus kit (Amersham Oxford, UK). Optimus software was used to quantitate Densitometric analyses (Optimus Corp).
3
Protein Extraction and Western Blot Analysis
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One hundred mg of adipose, liver, or intestine tissue was homogenized in a commercial PRO-PREP Protein Extraction Solution (Beyotime, China). Total protein lysates were fractionated by 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Immobilon™-P; Merck Millipore, USA). Afterwards, the membranes were blocked with 5% nonfat milk for 1 h at room temperature in TBST buffer (10 mM Tris, 150 mM NaCl, pH 7.6, and 0.1% Tween 20) and probed with primary antibodies overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody. The dilutions of primary and secondary antibodies have been described in Section 2.12 . Protein bands were developed using an enhanced chemiluminescence reagent (Merck Millipore). The blots were probed with the primary antibodies against GAPDH (Bioworld Technology, Inc., China), TLR4, IKB-α, NF-κB (Abcam, USA), AKT, phospho- (p-) AKT (Ser473), GSK3β, and p-GSK3β (Ser9) (Cell Signaling Technology, USA).
4
Quantitative Protein Analysis of Tissues
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Total proteins of tissues, mouse tumors and cells were extracted by using 150 µl PRO-PREP Protein Extraction Solution (iNtRON), which were measured by BCA protein assay kit (Beyotime Institute of Biotechnology). Protein (40 µg) was added to 10% SDS-PAGE and the separated protein was transferred onto polyvinylidene fluoride membranes (PVDF; EMD Millipore), which were blocked by 5% non-fat milk for 1 h. Then TBST (Boster) was used to wash the membranes for three times, which were incubated with primary antibodies overnight at 4°C, which were purchased from Abcam. The antibodies used in the western blot analysis are listed in Table II . After that, they were incubated with matched secondary antibodies (goat anti-rabbit, dilution 1:5,000, product code ab150077; goat anti-mouse, dilution 1:5,000; product code ab150113) for 1 h. Finally, the proteins were visualized and quantified by using Pierce ECL Western blot substrate (Thermo Fisher Scientific, Inc.) with ECL detection system (Thermo Fisher Scientific, Inc.) and proteins were normalized to β-actin. The gray value of the target band was analyzed by using Image Lab (version 3.1; Bio-Rad Laboratories, Inc.).
5
Protein Extraction and Western Blot Analysis
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Rat colonic tissues and IEC-6 cells were homogenized using a commercial Pro-Prep Protein Extraction Solution (Beyotime). Protein concentrations were determined using the BCA kit (Beyotime) following the provided instructions. Subsequently, 30 µg of protein sample was separated by 12% SDS-PAGE and transferred onto PVDF membranes (Millipore). After blocking with 5% BSA, the membranes were incubated overnight with primary antibodies, including anti-GPR41 (1:500, PA5-75521, Thermo Fisher Scientific), anti-GPR43 (1:200, PA5-111780, Thermo Fisher Scientific), anti-cleaved-caspase-3 (1:1,000, GTX86952, GeneTex), anti-β-actin (1:1,000, ab8227, Abcam) and anti-GAPDH (1:2,000, ab181602, Abcam). Following incubation with suitable secondary antibodies, protein bands were visualized using ECL detection reagents (Thermo Fisher Scientific). The results were presented as the ratio of target protein intensity to the intensity of GAPDH and β-actin loading reference band in the same lane.
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