Cells were grown to 70–80% confluency, harvested and lysed in Bicine/CHAPS buffer (ProteinSimple, San Jose, CA, USA) in the presence of protease/phosphatase inhibitors. Total protein concentrations were determined by Piece BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). A total of 20 μg proteins per well was loaded, transferred onto nitrocellulose membrane, and subsequently probed with xCT and GAPDH antibodies. Specific protein band intensity was quantified using ImageJ software (NIH).
Piece bca protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Piece BCA protein assay kit is a colorimetric assay for the quantitative determination of total protein concentration. The kit utilizes the bicinchoninic acid (BCA) method to detect and quantify the total protein present in a sample. The assay produces a purple-colored reaction product that exhibits a strong linear absorbance at 562 nm, allowing for the accurate measurement of protein levels.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Intercept blocking buffer, by LI COR (1 mentions)
Irdye 800cw anti rabbit antibody, by LI COR (1 mentions)
Las 300 imaging system, by Fujifilm (1 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti p stat3, by Cell Signaling Technology (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti six1, by Cell Signaling Technology (1 mentions)
Anti mmp9, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
11 protocols using piece bca protein assay kit
1
Protein Expression Quantification
2
Protein Extraction from Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
De-identified human tumor samples of patients enrolled on a Phase II riluzole monotherapy trial were obtained from Biospecimen Facility Core at Rutgers Cancer Institute of New Jersey by the approval of Rutgers Institutional Review Board (IRB). Informed, signed consent for the use of the tumor samples was obtained from all the patients. Each tumor sample was processed for total protein extraction. Briefly, frozen tissue was grind with mortar and pestle in the presence of liquid nitrogen and the resulting tissue powder was subjected to protein extractions by incubating with Bicine/CHAPS extraction buffer (ProteinSimple, San Jose, CA, USA) on a shaker for 1 h at 4 °C. The samples were then centrifuged at 13,000 × g for 10 min and the supernatants were collected. Protein concentration was determined by Piece BCA protein assay kit (Pierce Biotechnology, Rockford, IL USA). 20 μg of total proteins per well were resolved by 4%-12% gradient SDS-PAGE.
3
KCNJ15 Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein from tissues or cells was extracted using cold RIPA lysis buffer (150 mM NaCl, 50 mM Tris-base, 5 mM EDTA, 1% NP-40, and 0.25% deoxycholate, pH 7.4) with protease inhibitors (Thermo Fisher Scientific). The obtained protein was relatively quantified using a Piece BCA Protein assay kit (Thermo Fisher Scientific) and then stored in a –80°C freezer. The extracted protein samples (20 µg) were electrophoresed by SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat skim milk in 50 mM Tris–HCl, 50 mM NaCl, and 0.1% Tween-20 (TBST) at room temperature for 2 hours and then incubated with primary antibodies including mouse polyclonal anti-KCNJ15 antibodies (1:3,000 ratio; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rat polyclonal anti-GAPDH antibodies (1:8,000; Abcam, Cambridge, UK) at 4°C overnight. After the membranes were washed with 1× TBST buffer three times, horseradish peroxidase-labelled goat anti-rabbit antibodies (1:8,000; Abcam) and goat anti-mouse antibodies (1:8,000; Abcam) were added as secondary antibodies at room temperature with gentle shaking for 1 hour.
4
Western Blot Quantification of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentrations were examined using a Piece BCA protein assay kit (Thermo Fisher Scientific). A total of 10µg of the protein of each sample was loaded in a lane of the precast NUPAGE 4–12% Bis-Tris gels (Thermo Fisher Scientific). Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane for two hours at 400 mA. The membrane was blocked for 30 min using an Intercept blocking buffer (LI-COR) and the primary antibodies against TH (1:2500, Abcam, Cambridge, UK) or GAPDH (1:10,000, Millipore, Burlington, MA, USA) were applied and incubated overnight at 4 °C. After being washed with 1x PBST (0.1% Tween 20 in PBS) 4 times, the membrane was incubated with the fluorescent conjugated secondary antibodies (IRDye680RD anti-mouse antibody [1:20,000] or IRDye800CW anti-rabbit antibody [1:30,000], LI-COR, Lincoln, NE, USA) for one hour at room temperature. Membranes were scanned using an Odyssey Fc Imaging system (Millennium Science, Mulgrave, VIC, Australia). The intensity of specific bands for TH or GAPDH and their relative expression were quantified using Odyssey software.
5
Whole Cell Lysate Protein Quantification
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein lysate was harvested as previously described.21 Protein concentration was determined by Piece BCA protein Assay kit (Thermo Fisher Scientific) and was separated by 10% SDS‐PAGE and transblotted onto polyvinylidene difluoride membranes (PVDF). Next, PVDF membranes were blocked using 5% not‐fat milk in tris‐buffered saline for 1 hour at room temperature, and membranes were incubated with primary antibodies at 4°C overnight. After washed, the membranes were incubated with the appropriate secondary antibodies for 1 hour at room temperature. Finally, an enhanced chemiluminescence ECL Detection Kit was used to evaluate the bands, and a transilluminator was used to examine the intensity of the image. The primary antibodies included anti‐Six1, anti‐MMP‐9, anti‐p‐STAT3, anti‐STAT3, anti‐p65 and anti‐β‐actin (Cell signaling Technology, MA, USA).
7
Western Blot Analysis of CKAP4 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cultured cells were harvested and then lysed in ice-cold RIPA lysis buffer containing a proteinase inhibitor. Protein concentrations were evaluated using a Piece BCA Protein Assay Kit (Thermo Fisher Scientific). A volume of extract equivalent to 15 µg of total protein was separated in 12% acrylamide SDS-PAGE. The proteins were transferred to a methanol-activated polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA) after electrophoresis. Membranes were blocked with 5% nonfat dry milk for 1 hour, then incubated with primary antibody (CKAP4, 16686-1-AP, 1:1,000; Proteintech, Wuhan, China) overnight at 4°C. Afterward, the membranes were probed with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, Cambridge, MA, USA). The immune complexes were visualized using an electrogenerated chemiluminescence kit. Protein levels were normalized to the internal control, β-actin.
8
Quantifying Tubulin Polymerization Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantification of cellular levels of polymerized and soluble tubulin was performed by western blotting. Total protein was isolated from cells using radioimmunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, MO) followed by centrifugation ot separate the soluble (monomeric) tubulin fraction from the insoluble polymerized fraction. Protein concentration was quantified using the bicinchoninic acid method with the Piece BCA Protein Assay Kit (Thermo Fisher). Soluble and polymerized protein lysates were loaded onto 4–12% NuPAGE Bis-Tris gels (Invitrogen, Thermo Fisher), resolved by electrophoresis, and then transferred to PVDF membranes. Membranes were incubated with blocking buffer (5% BSA or 5% non-fat milk) for 30 minutes, and then probed with primary antibody against β-tubulin (Cell Signaling) overnight at 4 °C. Membranes were incubated with IRDye infrared fluorescent secondary antibodies (Li-Cor) and bands were detected and quantified using the Odyssey CLx Infrared Imaging System (Li-Cor).
9
Protein Expression Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from the cells using RIPA lysis buffer containing protease inhibitors (Beyotime) and quantified using the Piece BCA Protein Assay Kit (Thermo Scientific). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on to polyvinylidene fluoride membranes after extraction. Next, we blocked the membrane with 5% nonfat milk in 0.1% PBST and incubated the membrane with the indicated antibodies. The following primary antibodies were used: β-Actin (A5441, Sigma), Akt (#4685, CST), p-Akt (Ser473) (#4060, CST), p-p70 S6K (Thr389) (#9234, CST), p-mTOR (Ser2448) (#5536, CST), vimentin (#5741, CST), Slug (#9585, CST), and Snail (#3879, CST). Detection of the antigen-antibody complex on the membrane was achieved using enhanced chemiluminescence (Bio-Rad). Blots were quantified using Image J software.
10
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed with RIPA buffer (Beyotime, Shanghai, China) containing 1X PMSF and a protease inhibitor cocktail. After centrifugation at 4 °C and 12,000× g for 15 min, the concentration of proteins in the supernatant was determined using a Piece BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The proteins were separated using 10% sodium dodecyl-sulfate-polyacrylamide gels. Thereafter, they were transferred onto a PVDF membrane using a transfer apparatus, and after blocking with 5% skimmed milk, the membrane was incubated with the primary antibody overnight at 4 °C. The primary antibodies, rabbit anti-EGFR, rabbit anti-phospho-EGFR, rabbit anti-FASN, rabbit anti-LIPIN1, rabbit anti-Insig1, rabbit anti-SCD1, rabbit anti-SREBP1, rabbit anti-pAMPK, rabbit anti-p-AKT, rabbit anti-p-PI3K, and mouse anti-GAPDH, were purchased from Cell Signaling Technology (Danvers, MA, USA). Further, rabbit anti-Srebf1 was purchased from Abcam (Cambridge, MA, USA), while goat anti-rabbit IgG and goat anti-mouse secondary antibodies were purchased from Proteintech (Rosemont, IL, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!