After lysing the cells, the protein concentration was assessed utilizing the BCA Quantitation Kit (Thermo). Subsequently, an equal amount of protein was resolved by 6% SDS-PAGE and transferred onto a nitrocellulose filter membrane. At room temperature, the membrane was treated in 3% skim milk for 2 h to block non-specific binding. In this study, due to multiple antibodies incubated at the same time will interfere with each other, resulting in mixed bands, the membrane is cut and underwent overnight incubation at 4 °C with specific primary antibodies respectively: BAZ2A (ab290639, Abcam, Cambridge, MA, USA), GADPH (ab8245, Abcam), BAX (ab32503,Abcam), Snail (MA5-14801, Thermo), p53 (GTX34938, GeneTex, TX, USA), Bcl-2 (GTX100064, GeneTex), Vimentin (GTX40346,GeneTex), E-cadherin (CSB-RA576116A0HU, CUSABIO, Wuhan, China), and N-cadherin (CSB-RA243509A0HU, CUSABIO). The membrane was subjected to 1 h of incubation with the corresponding secondary antibody (CSB-PA564648/CSB-PA573747, CUSABIO). ECL chemiluminescence substrate (PerkinElmer, Waltham, MA, USA) was used to detect bands.
Bca quantitation kit
Manufactured by Thermo Fisher Scientific
The BCA Quantitation Kit is a reagent-based assay used to determine the concentration of protein samples. It relies on the bicinchoninic acid (BCA) method to quantify the total protein content in a sample by measuring the color change resulting from the reduction of copper ions. The kit provides a simple, colorimetric procedure for the quantitative determination of total protein concentration.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Cusabio (1 mentions)
Ab32503, by Abcam (1 mentions)
Ma5 14801, by Thermo Fisher Scientific (1 mentions)
Ab8245, by Abcam (1 mentions)
Bcl 2, by GeneTex (1 mentions)
Vimentin, by GeneTex (1 mentions)
Wbkls0100, by Merck Group (1 mentions)
Hatf00010, by Merck Group (1 mentions)
Byl40422, by BD (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Tris buffered saline (tbs), by Beyotime (1 mentions)
Tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l, by Beyotime (1 mentions)
4 protocols using bca quantitation kit
1
Protein Expression Analysis by Western Blot
2
Protein Extraction and Western Blot Analysis
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Proteins in tissues or cells were extracted by RIPA buffer (containing protease and phosphatase inhibitor; Solarbio, R0010) and quanti ed by BCA Quantitation Kit (Thermo, PICPI23223). About 25 μg of protein was isolated by 10% SDS-PAGE (Shanghai JRDUN Biotechnology) and then transferred to a polyvinylidene uoride (PVDF) membrane (Millipore, HATF00010) by electro-blotting. The cells were blocked for 1 h in 5% skim milk (BD Biosciences, BYL40422), and incubated for 2h at room temperature.
The cells were washed 5 times with PBST and incubated with secondary antibody (1:1000, Beyotime) at 37°C for 1 h in the dark. The chemiluminescence detection reagent (Millipore, WBKLS0100) was added and incubated for 5 min in the dark and then exposed on the ECL (Tanon, Tanon-5200). Relative protein levels were calculated using Image J software.
The cells were washed 5 times with PBST and incubated with secondary antibody (1:1000, Beyotime) at 37°C for 1 h in the dark. The chemiluminescence detection reagent (Millipore, WBKLS0100) was added and incubated for 5 min in the dark and then exposed on the ECL (Tanon, Tanon-5200). Relative protein levels were calculated using Image J software.
3
Intestinal Protein Expression Analysis
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The protein expressions of β-defensin 2, MUC2, tight junction proteins: ZO-1 and Occludin in the intestine were determined by western blot. Total protein extraction was performed using T-PER Tissue Protein Extraction Reagent (Thermo Pierce, 78510), protein quantification was then performed using the BCA Quantitation Kit. After the process of SDS-page electrophoresis analysis, transferred membranes, T-TBS (containing 5% non-fat dry milk or BSA) was added to the membrane and blocked at room temperature for 1 h. Antibody (1: 100) was added and incubated overnight at 4°C, then washing the membrane. Secondary antibody [Goat anti-Mouse IgG (H + L)] was added and incubated at room temperature for 1 h, and then washed the membrane. SuperSignal® West Dura Extended Duration Substrate was used for Western blot detection. The optical density of the bands was analyzed using Image J software. The β-actin was used as an internal control, which exhibited no difference between the groups. The relative abundance of each target protein was expressed as the ratio of target protein/β-actin protein.
4
Quantifying Intestinal Mucin and Tight Junctions
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The protein expression of mucin 2 (MUC-2) and tight junction proteins, including claudin-1, in the jejunum tissue was determined by Western blotting. Total protein extraction was performed using Tissue Protein Extraction Reagent (Thermo Pierce, 78510), and protein quantification was then performed using the BCA Quantitation Kit. After SDS-PAGE and membrane transfer, Tris-buffered saline (containing 5 % non-fat dry milk or bovine serum albumin) (Beyotime Biotechnology) was added to the membrane for blocking at room temperature for 1 h. The antibody (1:100) (Beyotime Biotechnology) was then added and incubated overnight at 4°C, followed by washing the membrane. Secondary antibody (goat anti-Mouse IgG (H + L)) (Beyotime Biotechnology) was added and incubated at room temperature for 1 h and then washed. SuperSignal® West Dura Extended Duration Substrate was used for Western blot detection. The optical densities of the bands were analysed using Image J software. β-Actin was used as an internal control and was found to exhibit no differences between groups. The relative abundance of each target protein was expressed as the ratio of target protein:β-actin.
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