Cells were seeded in 10 cm dishes 24 hours prior to treatment and incubated with indicated compounds for 1 hour. Cells were washed with phosphate-buffered saline (PBS) and lysed with NP-40 lysis buffer (1% NP40, 150 mM NaCl, and 25 mM Tris, pH 8.0) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Concentration of protein was determined using Lowry assays (Bio-Rad, Hercules, CA) and equal amount of whole cell protein lysate was loaded in each lane and resolved using 4-12% gradient Bis-Tris gel (Invitrogen, CA). Proteins were transferred to 0.2-0.45μm nitrocellulose membrane (Invitrogen, CA). Membranes were incubated overnight at 4 °C with primary antibodies after blocking, followed by incubation with appropriate HRP-conjugated secondary antibody at room temperature for one hour. ECL-Plus was used to detect the activity of peroxidase according to the manufacturer's protocol (Amersham Pharmacia, Uppsala, Sweden). Antibodies raised against phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), pAKT(S473), phospho-p70 S6K and total ERK, AKT antibodies were purchased from Cell Signaling Technology (Beverly MA, USA), and anti-beta actin (conjugated with HRP) was purchased from Abcam (Cambridge, MA, USA). Secondary HRP antibodies were purchased from Jackson ImmunoResearch (St. Louis MO, USA).
Lowry assay
Manufactured by Bio-Rad
Sourced in United States, Germany, United Kingdom
The Lowry assay is a colorimetric method used to determine the total protein concentration in a sample. It measures the absorbance of a color complex formed between copper ions and the aromatic amino acids in the protein, which is proportional to the protein concentration.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop, by Roche (7 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Ecl plu, by Cytiva (3 mentions)
0.2 μm nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions)
Bis tris gradient gel, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Laemmli sample buffer, by Bio-Rad (3 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Ecl plus reagent, by GE Healthcare (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Lysis buffer, by Thermo Fisher Scientific (2 mentions)
Victor 1420 multilabel counter, by PerkinElmer (2 mentions)
Complete mini edta free tablet, by Roche (2 mentions)
Near infrared fluorescent secondary antibodies, by LI COR (2 mentions)
0.45 μm nitrocellulose membrane, by Bio-Rad (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Exoquick kit, by System Biosciences (2 mentions)
92 protocols using lowry assay
1
Western Blot Analysis of Signaling Pathways
2
Western Blot Analysis of Tumor Cell Lysates
3
Western Blot Analysis of Tumor Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor samples were collected and snap-frozen in liquid nitrogen. Frozen tumor tissues were homogenized in ice-cold RIPA buffer supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche, Basel, Switzerland) and phosphatase inhibitors (PhosSTOP, Roche, Basel, Switzerland). Concentration of protein was determined using Lowry assays (Bio-Rad, Hercules, CA) and equal amount of whole tissue protein lysate was loaded in each lane and resolved using 4–12% gradient Bis-Tris gel (Invitrogen, CA). Proteins were transferred to 0.2 μm nitrocellulose membrane (Invitrogen, CA). Membranes were incubated overnight at 4°C with primary antibodies after blocking, followed by incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for one hour. ECL-Plus was used to detect the activity of peroxidase according to the manufacturer's protocol (Amersham Pharmacia, Uppsala, Sweden). The films were scanned using grayscale mode after their development.
4
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were homogenized and lysed with RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Similarly, cells were lysed with RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Protein concentrations of whole-cell lysates were determined using Lowry assays (Bio-Rad). 20 μg of total protein were prepared in LDS sample buffer (Invitrogen), separated on denaturing Bis-Tris gel (Invitrogen), and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked in 5% milk in 0.1% Tween 20 Tris-buffered saline (TBST) and subsequently incubated with primary antibodies against phospho-p44/42 MAPK (pErk1/2; Thr202/Tyr204), total ERK, RasG12D, and P53 (Cell Signaling Technology) in TBST overnight at 4°C, or with β Actin HRP-conjugated for 1 hour at room temperature. Secondary antibodies were purchased from Jackson ImmunoResearch. ECL-Plus substrate (Bio-Rad) and Bio-Rad ChemiDoc MP imager were used according to the manufacturer’s recommendations.
5
Analysis of Protein Expression in QM5 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Multi-well plates containing subconfluent QM5 cells were transfected with plasmid DNA using Lipofectamine LTX (Life Technologies) according to manufacturer's instructions. For western blotting, cells were lysed at 8 h post-transfection with RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% IGEPAL) containing 1 μM protease inhibitors (aprotinin, pepstatin, and leupeptin) for 45 min on ice. Equivalent protein loads, as determined by Lowry assays (Biorad), were analyzed by SDS-PAGE (15% acrylamide) and western blotting using anti-p14 antiserum (1∶20,000) or anti-actin antibodies (1∶2,500) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1∶10,000). For the endoglycosidase H (endo H) assays, cells were lysed at 24 h post-transfection, and prior to SDS-PAGE lysates were treated at 37°C for 2 h with endo H or N-glycosidase F (PNGase F) according to manufacturer's specifications (New England Biolabs). Membranes were developed using ECL-plus reagent (GE Healthcare) and imaged on a Typhoon 9410 variable mode imager (Amersham) or a Kodak 4000 mm Pro CCD imager.
6
Western Blot Analysis of AKT Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells treated by indicated conditions were pelleted by centrifugation and rinsed with PBS. The cell pellets were then lysed in RIPA buffer. DNA in the lysate was sheared by a sonicator. Concentration of protein was determined using Lowry assays (Bio-Rad). Protein extracts were loaded onto SDS-PAGE and were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.). The membranes were analyzed by specific primary (p-AKT, AKT, POSTN and GAPDH) and secondary antibodies. The signals were developed using an ECL chemiluminescence kit (Amersham Biosciences, UK).
7
Immunoblotting of Tumor Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor tissue from two individual animals from each of the treatment groups were collected for immunoblotting studies. Tumor tissues were snap-frozen in liquid nitrogen, and stored at − 80°C. Tissues were then homogenized in NP-40 lysis buffer supplemented with protease inhibitors and phosphatase inhibitors. Concentration of protein was determined using Lowry assays (Bio-Rad, Hercules, CA). Equal amount of protein were loaded in each lane and resolved by 4% to 12% gradient Bis-Tris gel (Invitrogen, CA). Proteins were transferred to 0.2 μm nitrocellulose membrane (Invitrogen, CA). Membranes were incubated overnight at 4°C with primary antibodies after blocking, followed by incubation with appropriate HRP-conjugated secondary antibody at room temperature for one hour. ECL-Plus was used to detect the activity of peroxidase according to the manufacturer’s protocol (Amersham Pharmacia, Sweden). Antibodies raised against PARP-1, pH2A.X(Ser139) were purchased from Cell Signaling Technology (Beverly, MA). Antibody raised against PAR was purchased from Trevigen (Gaithersburg, MD). HRP conjugated beta-actin was purchased from Abcam (Cambridge, MA).
8
Evaluating Protein Expression in Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cancer cells were treated with control vehicle, metformin, carboplatin, or a combination for 48 h, and the cells were pelleted by centrifugation and rinsed with PBS. The cell pellets were then lysed in RIPA buffer followed by sonication. Lowry assays (Bio-Rad) were performed to determine the protein concentration. Equal amounts of protein were loaded in each lane and resolved by 10 to 12% gradient Bis-Tris gels. All Western blot analyses were performed using whole-cell lysates prepared as described above. SDS-PAGE and Western blotting were performed using standard methods. The protein band intensities were quantified using ImageJ analysis by determining the relative intensity for each experimental band and normalizing its absolute intensity to that of the control.
9
Analyzing p14 protein expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 8 hpt, QM5 cells were lysed with RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% IGEPAL) containing 1 μM protease inhibitors (aprotinin, pepstatin, and leupeptin) for 45 min on ice. Equivalent protein loads, as determined by Lowry assays (Bio-Rad, Hercules, CA), were analyzed by SDS–PAGE (15% acrylamide) and Western blotting using anti-p14 antiserum (1:10,000) or antibodies against actin (1:2500) or the myc epitope tag (1:2500) and a HRP-conjugated goat anti-rabbit secondary antibody (1:10,000). For the endo H assays, cells were lysed at 24 hpt, and before SDS–PAGE lysates were treated at 37˚C for 2 h with endo H or PNGase F according to the manufacturer's specifications (New England BioLabs, Ipswich, MA). Membranes were developed using ECL-Plus reagent (GE Healthcare, Little Chalfont, UK) and imaged on a Typhoon 9410 variable-mode imager (GE Healthcare) or a Kodak 4000-mm Pro CCD imager. Blots were quantified by ImageJ (National Institutes of Health, Bethesda, MD) and results reported as band intensity relative to authentic p14.
10
Multicolor Protein and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were prepared in RIPA buffer and quantified by Lowry assay (BioRad). Western blots were probed with antibodies against: TetR (rtTA3) (1∶1000, mouse monoclonal Clone 9G9, Clontech #631131), GFP (1∶2000, chicken polyclonal, Abcam #ab13970), tRFP (mKate2) (1∶2000, rabbit polyclonal, Evrogen #B00201) and β-Actin-HRP (1∶5000, mouse monoclonal AC15 clone, Sigma #A3854). RNA was prepared from sorted cells by Trizol extraction and column purification. cDNA was prepared from 1 µg total RNA using Taqman reverse transcription kit (Applied Biosystems, #N808-0234) with random hexamers. Quantitative PCR detection was performed using SYBR green reagents (Applied Biosystems) using primers specific to rtTA3 : F: 5′-CAATGGTGTCGGTATCGAAG-3′ , R: 5′-CTTGTTCTTCACGTGCCAGT-3′ ; mKate2 : F: GGTGAGCGAGCTGATTAAGG-3′ and R: 5′-TTTTGCTGCCGTACATGAAG-3′ ; and GFP : F: 5′-ATCGACTTCAAGGAGGACGGCA-3′ and R: 5′-CGTTCTTCTGCTTGTCGGCCAT-3′ .
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!