Proteins were extracted from uterine tissues with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1 mM Na3VO3, 1% sodium deoxycholate, 1% Triton X-100 and 0.1% SDS). A complete protease inhibitor (Roche Applied Science, Upper Bavaria, Germany) was added into each sample to prevent protein degradation. Protein concentration was measured with BCA reagent kit (Applygen, Beijing, China). Samples were electrophoretically separated on 10% PAGE and electrically transferred onto PVDF membranes. After blocking with 5% non-fat milk containing 0.5% Tween 20 for 1 h, the membrane was incubated with rabbit anti-total STAT3 antibody (1:1000; #9132, Cell Signaling Technology) or rabbit anti-phosphorylated (Tyr 705) STAT3 antibody (1:1000; #9131, Cell Signaling Technology) overnight at 4°C. The membrane was then incubated with HRP-conjugated secondary antibody (1:5000) for 1 h. Signals were analyzed by ECL Chemiluminescent kit (Amersham Pharmacia Biotech, IL USA). Tubulin was used as a loading control.
Bca reagent kit
Manufactured by Applygen
Sourced in China
The BCA reagent kit is a laboratory product designed for protein quantification. It utilizes the bicinchoninic acid (BCA) assay method to determine the total protein concentration in a sample. The kit provides the necessary reagents and instructions to perform the BCA assay, which is a colorimetric detection technique.
Automatically generated - may contain errors
Lab products found in correlation
Ecl chemiluminescent kit, by Cytiva (2 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Protease inhibitor, by Selleck Chemicals (1 mentions)
Anti lc3b, by Merck Group (1 mentions)
Anti pink1, by Abcam (1 mentions)
Anti tom20, by Abcam (1 mentions)
Anti α actin, by Abcam (1 mentions)
Anti p62 sqstm1, by Merck Group (1 mentions)
Ecl detection reagent, by Applygen (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Enhance chemiluminescence kit, by Cytiva (1 mentions)
Vimentin, by Proteintech (1 mentions)
Non flat milk, by Sangon (1 mentions)
T stat3, by Cell Signaling Technology (1 mentions)
β tubulin, by Proteintech (1 mentions)
Ecl kit, by Sangon (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
8 protocols using bca reagent kit
1
Quantifying Protein Expression in Uterine Tissues
2
Apoptosis Protein Expression Analysis
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The protein expression of Bcl-2, Bax, caspase-3 and cytochrome C was measured by Western blot. The internal control was β-actin. After the cells were treated with gracillin for 24 h, the total protein of each group was extracted by a RIPA buffer (R0010; Solarbio, China) with 1% phenylmethylsulfonyl fluoride (PMSF). Then the concentration of the protein was detected by a BCA reagent kit (P1511; Applygen, China). The proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and electro-transferred onto a polyvinylidene fluoride membrane. The membrane was blocked in 5% nonfat dry milk (prepared by 1 × Tris-buffer saline and 0.05% Tween 20%) for 2 h and then incubated with the primary antibody (Applygen, Beijing, China) for 12 h at 4 °C. The membrane was rinsed and incubated at room temperature for 2 h with Horizontal shaker (Applygen, Beijing, China). Finally, the membrane was rinsed again and incubated with the chemically luminescent solution for exposure. Protein intensity was measured using Image Lab software.
3
Evaluation of RFA Treatment on Protein Levels
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786-O cells in logarithmic phase were treated with 0 μmol/L, 10 μmol/L, 20 μmol/L, 40 μmol/L RFA for 24 h. The total proteins from each group were extracted by a RIPA buffer containing 1% PMSF (R0010; solarbio, China) and the total protein concentration was detected by a BCA reagent kit (P1511; Applygen, China). The proteins were separated by 12% SDS-PAGE and electrotransferred to the PVDF membrane. After sealing with 5% skimmed milk for 2 h, the membrane was incubated with specific primary antibody at 4 °C for 12 h, washed with TBST for three times, 5 min each time, and incubated with horseradish peroxidase-labeled secondary antibody for 1 h. Finally, the secondary antibody that did not bind to the primary antibody was washed by the above method and incubated with chemiluminescence solution to expose the strip in the dark room. The optical density of protein bands was analyzed by gel imaging analysis system.
4
Western Blot Analysis of Autophagy Markers
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Heart tissues or cells were collected and lysed with RIPA buffer (Beyotime, P0013C) containing protease inhibitors (Selleck, B15002). The samples (20 µl) were used to determine the total protein concentrations with BCA reagent kit (Applygen, P1511-1). Protein samples were heat-denatured for 5 min at 95°C with the sample loading buffer, and 20 µg of total protein per sample were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, IPVH00010). The membranes were blocked with 5% BSA for 30 min and incubated overnight at 4°C shaking with primary antibodies. The membranes were then washed in Tris-buffered saline (TBS, pH 7.6) with 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies (1:4000, ZSGB-BIO, ZB2301) for 2 h at room temperature. Bands were visualized using ECL Detection Reagent (Applygen, P1050), and quantify analysis was performed using Gel-Pro Analyzer System. The primary antibodies used were presented as follow: anti-LC3B (1:5000, Sigma, L7543); anti-p62/SQSTM1 (1:5000, Sigma, P0067); anti-PINK1 (1:1000, Abcam, ab23707); anti-TOM20 (1:1000, Abcam, ab78547); anti-α-Actin (1:5000, Abcam, ab156302).
5
Western Blot Protein Analysis
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Tissues were homogenized in lysis buffer. The concentration of protein was measured with the BCA reagent kit (Applygen). Protein samples were separated by electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20 for 1 h at room temperature and then incubated overnight at 4 °C with primary antibody. After three washes in 5% milk in TBS-T, membranes were incubated with horseradish peroxidase (HRP)-conjuncted secondary antibody for 1 h at room temperature. The signal was developed with the enhance chemiluminescence kit (Amersham Biosciences). Band intensities were analyzed with the ImageJ software. Uncropped and unprocessed scans of the blots are presented in SI Appendix, Fig. S17 . Primary antibodies with detailed information are listed in SI Appendix, Table S5 .
6
Uterine Protein Extraction for Early Pregnancy
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From day 3 to 5 of pregnancy, the uterine flushing fluids were collected by flushing the uterine horns with 200 µl of saline water. The liquids were centrifuged to discard hemocytes, castoff cells and embryos; the supernatant was collected; and the proteins were extracted by TCA-Acetone precipitation methods, as previously described [26] (link). The extracted protein concentration was detected by a BCA reagent kit (Applygen, Beijing, China), diluted to a unified concentration and used for western blot analysis.
7
Protein Expression Analysis of Stromal Cells
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As described previously (Lin et al. 2018) . Proteins were extracted from cultured stromal cells with lysis buffer. The BCA reagent kit (Applygen, Beijing, China) was used to measure the concentration of proteins. The protein samples were separated by 12% SDS-PAGE gel and transferred onto PVDF membranes (Millipore). PVDF membranes were blocked with 5% non-flat milk (Sangon, Shanghai, China) and followed by probing with the corresponding antibodies for CYPA (final concentration: 1 µg/mL; Abcam), p-Stat3 (final concentration: 12 ng/mL; Cell signaling), T-Stat3 (final concentration: 24 ng/mL; Cell signaling), Vimentin (final concentration: 0.27 µg/mL; Proteintech), Cytokeratin 18 (CK18, final concentration: 0.3 µg/mL; Proteintech), β-Tubulin (final concentration: 0.1 µg/mL; Proteintech) and Gapdh (final concentration: 0.2 µg/mL; Santa Cruz) overnight at 4°C. After washing, the matched secondary antibodies conjugated with HRP (final concentration: 0.1 µg/mL; Elabscience) were incubated PVDF membranes. Signals were developed with the ECL kit (Sangon, Shanghai, China).
8
Endometrial Tissue Protein Extraction and Analysis
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Whole endometrial tissue lysate was prepared and homogenized in lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, and 0.25% sodium deoxycholate). Protein concentration was measured and adjusted by using the BCA Reagent kit (Applygen, Beijing, China). Samples were run on a 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in 5% nonfat dry milk in TBST (0.1% Tween 20 in TBS) for 1 h, membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-human telomerase (ab68781, Abcam, Cambridge, UK), ER alpha (sc-7207, Santa Cruz, CA, USA) or anti-human Gapdh (sc-25778, Santa Cruz). After three washes in TBST, membranes were incubated with secondary antibodies conjugated with horseradish perioxidase for 1 h at about 25°C. The signals were developed with the ECL Chemiluminescent kit (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). Films were scanned using a flat-bed scanner. The intensities of the bands representing ER alpha and TERT were evaluated using the Image J analysis software (Rawak Software, Inc., Germany).
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