The hBMSCs on the membranes and in the control were lysed after osteogenic differentiation for 7 days with Cell Lytic buffer for 30 min. These lysates were sonicated for 10 s and centrifuged at 14,000 rpm for 15 min at 4°C. The protein concentrations were determined using a BCA protein assay kit (Roche, Rockford, IL, USA). Equal amounts of the lysates were loaded onto a 5% stacking and 10% resolving SDS-PAGE gel. After electrophoresis, the proteins were transferred to PVDF (polyvinylidene fluoride) membranes and reacted with Runx2 (1:1,000, Abcam) antibody. For measurement, horseradish peroxidase-conjugated secondary antibodies (1:1,000; Jackson ImmunoRes, West Grove, PA, USA) were used, followed by enhanced chemiluminescence (ECL) Plus Western Blotting Detection System (GE Health-care UK Ltd, Little Chalfont, UK). The immunoreactive bands formed were quantified by scanning densitometry software (ImageJ; National Institutes of Health, Bethesda, MD, USA). Protein expression levels were normalized to those of a housekeeping gene, β-actin (1:1,000 dilution with 1% bovine serum albumin in 0.01 M PBS; Santa Cruz Biotechnology Inc., Dallas, TX, USA). All Western blotting analyses were repeated three times under the same conditions.
Bca protein assay kit
Manufactured by Roche
Sourced in Switzerland, United States
The BCA protein assay kit is a colorimetric detection and quantitation assay used for the determination of total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reaction, where proteins reduce Cu2+ to Cu+ in an alkaline medium, and the purple-colored reaction product is measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Santa Cruz Biotechnology (1 mentions)
Runx2, by Abcam (1 mentions)
Ecl plus western blotting detection system, by GE Healthcare (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ldh activity assay kit, by Abcam (1 mentions)
Enhanced chemiluminescence assay, by Merck Group (1 mentions)
Peroxidase conjugated goat anti rabbit antibody, by Merck Group (1 mentions)
Anti mbp, by AnaSpec (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Complete mini, by Roche (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Goat anti mouse or rabbit igg, by Abcam (1 mentions)
Ecl western blotting substrate kit, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
β actin, by Abcam (1 mentions)
Snail antibody, by Cell Signaling Technology (1 mentions)
Gapdh antibody, by CWBIO (1 mentions)
Mmp 2 antibody, by Affinity Biosciences (1 mentions)
Srebp1 antibody, by Proteintech (1 mentions)
12 protocols using bca protein assay kit
1
Quantifying Osteogenic Runx2 Expression
2
Colorimetric LDH Activity Assay
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LDH Activity Assay Kit (#K726-500, Biovision, Tucson, AZ, USA) was used to determine the intracellular LDH activity. In this test, LDH reduces NAD to NADH, which interacts with a specific probe to produce a color (λmax = 450 nm), which is then detected by colorimetric assay. Results were expressed as percentage of LDH activity normalized to protein concentration, which were measured by BCA protein assay kit (Roche, USA).
3
Western Blot Analysis of MBP Expression
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Western blot analysis was performed to test the expression of MBP protein. Ten larvae were lysed in buffer with protease inhibitors (Complete Mini, Roche, Mannheim, Germany), and the protein concentrations were quantified with a BCA Protein Assay Kit (Complete Mini, Roche, Mannheim, Germany). The proteins were separated in a 10% SDS-PAGE gel and were transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was then blocked in 5% nonfat dry milk in PBS for 1 hour and incubated with anti-MBP (1∶500; Anaspec, Fermont, CA) and anti-GAPDH (1∶3,000; Millipore). The blots were rinsed with PBS and incubated with peroxidase-conjugated goat anti-rabbit antibodies (1∶5,000; Sigma-Aldrich, St. Louis, MO) for 1 hour. The bound antibody was visualized using an enhanced chemiluminescence assay (Millipore).
4
TRIM44 and POSTN Protein Expression Analysis
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As previously described [21 (link)], LN229 and U251 cells were harvested and lysed by RIPA lysis buffer (Beyotime). The protein was quantified by BCA Protein Assay Kit, and then 20 μg protein samples were electrophoresed by SDS-PAGE gel followed by transferring onto a PVDF membrane (Roche, Basel, Switzerland). After that, the membrane was incubated with nonfat milk for 2 h and then hatched with specific primary antibodies targeting TRIM44 (1:2,000, Abcam, Cambridge, MA, USA), POSTN (1:1,000, Abcam), or β-actin (1:1,000, Abcam) at 4°C overnight. After the membrane was cultured with Goat anti-Mouse or Rabbit IgG (1:20,000, Abcam) for 1 h, ECL Western Blotting Substrate Kit (Biovision) was used to visualize the protein bands in the membrane
5
Protein Expression Analysis in Cervical Cancer
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First, the whole protein was extracted by lysing cervical cancer cells with RIPA buffer before measuring the protein concentration via a BCA protein assay kit (A8020‐5, Roche, Basel, Switzerland). The cells were dissolved with SDS‐PAGE loading buffer and transferred into PVDF membranes. Then, the cells were incubated with diluted primary antibodies at 4°C after blocking with 5% skim milk dissolved in TBST for 1 hour. Primary antibodies used include HSDL2 antibody, E‐cadherin antibody, Vimentin antibody, Snail antibody, Slug antibody (Cell Signaling Technology, Boston, USA), MMP‐2 antibody (Affinity, Cincinnati, USA), FASN antibody, ACSL1 antibody, SREBP1 antibody (Proteintech) and GAPDH antibody (CWBIO). Next, goat antimouse IgG conjugated to HRP was applied as secondary antibody for 1 hour at room temperature. The cells were shortly incubated by ECL detection reagent, then detected by Imager.
6
Quantitative Analysis of Protein Levels
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Cell extracts were prepared using ice-cold RIPA buffer supplemented with complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), and total protein was quantified by the BCA Protein Assay Kit following the instructions of manufacturers. Protein was separated by a 10% SDS–polyacrylamide gel electrophoresis and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were probed with anti-LPL (1:5000, Abcam, Cambridge, UK) or anti-β-actin (1:2000, Abcam) antibody. Horseradish peroxidase-labeled IgG (1:5000, Abcam) antibody was used as a secondary antibody. Protein bands were determined by ECL reagent (Amersham Bioscience, Munich, Germany) and the intensity of the bands was analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
7
Western Blot Analysis of Protein Signaling
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Total protein was extracted using an extraction kit (Beyotime, China) according to the manufacturer’s protocols, and the protein concentration was determined by a BCA protein assay kit (Roche, Basel, Switzerland). Protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (40 µg per lane), then transferred to polyvinylidene fluoride membranes. After blocking with 5% non-fat dried milk in Tris-buffered saline with Tween-20 for 1 hour, the membranes were incubated with the appropriate primary antibody. Antibodies against β-actin (1:1000), c-Jun N-terminal kinase (JNK; 1:1000), p38 mitogen-activated protein kinase (MAPK; 1:1000), extracellular signal-regulated kinase (ERK; 1:1000), phospho-JNK (1:500), phospho-p38MAPK (1:500), and phospho-ERK (1:500) were purchased from Cell Signaling Technology® (Danvers, MA, USA). They were then incubated with horseradish peroxidase-conjugated secondary antibody (Bioss, Beijing, China) and visualized using the ECL plus detection kit (Pierce Protein Research Products, Rockford, IL, USA). Protein expression was quantified by densitometry using a ChemiDoc XRSþ image analyzer (Bio-Rad, Hercules, CA, USA) with β-actin as an internal loading control.15 (link)
8
Western Blot Analysis of RFC2 and β-Tubulin
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The total proteins from HS683, SW1783, and HMC3 cells were extracted by using radioimmunoprecipitation (RIPA) lysis buffer (Sigma Aldrich, Cambridge, MA), and then quantified by using BCA Protein Assay Kit (Roche, Switzerland). Then, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), which were then incubated overnight at 4 °C with primary antibodies, including RFC2 (1:1000, 10410-1-AP, Proteintech, USA) and β-Tubulin (1:1000, 10094-1-AP, Proteintech, USA). Subsequently, the membranes were then incubated with the secondary antibody (1:2000, Beyotime, Shanghai, China) labeled with horseradish peroxidase for 1 h at room temperature. Then, the protein bands were visualized by using the electrochemiluminescence reagent kit (Millipore, USA).
9
Quantitative Western Blot Analysis
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Synovial tissues and FLS were mixed with RIPA lysate and grinded for 10-15 min, respectively. Samples were agitated on ice for 30 min and the supernatant was collected. The protein levels were quantified with a BCA protein assay kit (Roche, Basel, Switzerland). Then the protein samples were electrophoresed in SD-PAGE to separate protein bands. Proteins were transferred from gel onto PVDF membrane, blocked with 5% nonfat dry milk for 2 h. The membrane was incubated with the first antibody (1 : 1000) for overnight at 4°C, and then with the second antibody for 2 hours. Later bands were visualized by exposure to ECL method and the overall gray value of protein bands (average gray value area) was quantified, GAPDH as internal marker, namely, target protein gray value/internal reference overall gray value.
10
Western Blot Protein Analysis
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FLS were mixed with RIPA lysate (Beyotime Biotechnology) and ground for 10-15 min. Samples were agitated on ice for 30 min, and the supernatant was collected. The protein levels were quantified using a BCA protein assay kit (Roche, Basel, Switzerland). Then, the protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate protein bands. Proteins were transferred from the gel onto polyvinylidene fluoride membrane and blocked with 5% nonfat dry milk for 2 h. The membrane was incubated overnight with the primary antibody (1: 500) at 4°C and then with the secondary antibody for 2 h. The bands were visualized by the electrochemiluminescence method, and the overall gray values of protein bands (average gray value area) were quantified. At the same time, β-actin was used as an internal marker to compare the gray value of target protein in different groups.
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