Adult mouse tissue extracts were obtained after lysis with buffer containing 50 mM Tris–HCl pH 8.0, 170 mM NaCl, 1% NP40, 5 mM EDTA, 1 mM DTT and protease inhibitors (Complete mini; Roche diagnostics GmbH)). Protein concentration for lysates was determined using BCA reagent (Sigma-Aldrich), and equal amounts of protein (50 μg/lane) were heated to 95°C for 10 min in sample buffer, loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels, electrophoretically separated, and transferred to polyvinylidene difluoride membranes (Millipore Corporation, Bedford, Mass.). Membranes were blocked (Tris-buffered saline solution containing 5% nonfat dry milk and 0.05% Tween 20 [TBST]) and then incubated with the indicated primary antibodies at 4°C overnight. After being washed in TBST, the blots were incubated with immunoglobulin conjugated to horseradish peroxidase for 1 h at room temperature. After washing, the target proteins were detected with Super Signal chemiluminescent substrate (Pierce). The following primary antibodies were used: Anti-LZTFL1, SIGMA-ALDRICHTM, 1:2,000, Cat No: AV48390-100UL; GAPDH, Santa Cruz Biotechnology, 1:2,000, Cat No: SC-32233. Secondary antibodies include: Anti-Rabbit IgG, Jackson ImmunoResearch, 1:2,000, Cat No: 711166152; Anti-Mouse IgG, Vector Laboratories, 1:2,000, Cat No: DI-2488.
Bca reagent
Manufactured by Merck Group
Sourced in United States, United Kingdom
The BCA reagent is a colorimetric reagent used for the quantitative determination of total protein concentration. It is based on the bicinchoninic acid (BCA) method, which combines the reduction of Cu2+ to Cu+ by proteins in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by BCA. The resulting purple-colored reaction product exhibits a strong absorbance at 562 nm that is linear with increasing protein concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Complete mini, by Roche (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
Glass bead, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Sartorius (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
Total jnk, by Santa Cruz Biotechnology (1 mentions)
Total erk, by Santa Cruz Biotechnology (1 mentions)
Cdc25c, by Santa Cruz Biotechnology (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Total p38, by Santa Cruz Biotechnology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
7 protocols using bca reagent
1
Protein Extraction and Western Blot Analysis
2
Antibody Purification from Human Serum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human serum samples were diluted 1:5 in PBS before loading onto a 2 ml protein G column (protein G beads – A2S, gravity column, Thermo scientific), with the flow-through collected and reloaded 3 times. Bound antibody was eluted with 0.1 M glycine buffer (pH 3.0). The elution was neutralized by adding 20% v/v 1 M Tris–HCl (pH 7.3). Next, protein-containing fractions were pooled and buffer exchange into PBS was performed by centrifugal filtration with 50KD cutoff (Amicon, Merck). Antibodies concentration was determined using the BCA reagent (Sigma-Aldrich). Antibody purity was assessed by SDS-PAGE.
3
Western Blot Protein Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A western blot analysis was performed as previously described [20 (link)]. The protein quantity was determined using a bicinchoninic acid (BCA) reagent (Sigma-Aldrich). Twenty grams of protein extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to the polyvinylidene difluoride (PVDF) membrane (Amersham, Buckinghamshire, UK). The membrane was blocked in 5% fat-free milk in Tris-buffered saline with Tween 20 (TBST) (20 mM Tris-HCl, pH 7.6, containing 0.4% Tween 20) and was incubated with primary antibodies for 24 h. The membrane was further incubated for 1 h with horseradish peroxidase-conjugated secondary antibody. The proteins were visualized using an enhanced chemiluminescence (ECL) detection system (GE Healthcare, Little Chalfont, UK).
4
Yeast Cell Lysis and Protein Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After resuspending 0.2 ml of the yeast cell pellet in 0.4 ml TBS, 1 g glass beads (Sigma, Ronkonkoma, NY) were added to each tube. Cells were then disrupted by vortexing vigorously for 20 min at 4°C. At the end of vortexing, the tube was centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant (total lysate) was collected. The protein concentration of total lysate was determined by BCA reagent (Sigma).
5
Apoptosis and Cell Cycle Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMEM, Antibiotic Antimycotic solution, Trypsin EDTA, Dimethyl sulfoxide (DMSO), propidium iodide (PI), protease and phosphatase inhibitor cocktail, BCIP-NBT, BCA reagent, carbonyl cyanide m-chlorophenyl hydrazone (mClCCP; a mitochondrial uncoupler), 3,3′-dihexyloxacarbocyanine iodide (DiOC6), MTT, ERK inhibitor (U0126), p38 inhibitor (SB203580), Cisplatin (CDDP) were purchased from Sigma (St. Louis, Missouri, USA). Caspase-8 inhibitor and zVAD-fmk (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethyl-ketone) were from calbiochem, Germany. Foetal bovine serum was purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Antibodies Cyclin-A, Cyclin-E, CDK-2, Cyclin-B1, p21, p27, Bid, PARP, cleaved caspase-3, −8, −9 were purchased from Cell signaling technologies (MA, USA). Antibodies Bax, Bak, Bcl-xL, cMyc, GAPDH, pAKT (Ser 473), AKT, p53, pCDC2, CDC2, CDC25c, pP38, total P38, pJNK, total JNK, pERK1/2, total ERK were purchased from Santacruz biotechnology (Santa Cruz, CA, USA). MFX and CFX were obtained from Cipla (India).
6
Analytical Methods for Extracting Proteins and Polysaccharides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For each EPS extract, the amounts of proteins and polysaccharides were measured in analytical triplicates. Protein (PN) concentration was measured by the bicinchoninic acid (BCA) method (Smith et al., 1985 (link)). Twenty-five μL of EPS extract and 200 μL of BCA reagent (Sigma-Aldrich) were incubated in a 96-well microplate (15 min at 60°C) before measuring the optical density at 540 nm (microplate reader, Synergy Mx Biotek). Bovine serum albumin (BSA) with concentrations ranging from 0 to 800 mg L–1 was used as a standard. Polysaccharide (PS) concentration was measured by the anthrone method (Dreywood, 1946 (link)). One hundred μL of EPS extract and 200 μL of anthrone reagent (2% anthrone in 96% sulfuric acid) were incubated in a 96-well microplate (30 min at 60°C), cooled at room temperature for 10 min before measuring the optical density at 620 nm. Glucose with concentrations ranging from 0 to 100 mg L–1 was used as a standard.
7
Transfection of A549 Luciferase Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For transfection studies, A549 luciferase expressing cells (5 Â 10 4 ) were seeded into 24-well plates and cultured in 500 mL of cell medium with FBS at 10%. After 24 h, the culture medium was replaced with 0.5 mL of fresh serum-free DMEM and treated with siRNA/OBAE polyplexes containing 1 mg of siRNA/well. After 4 h of incubation, cells were washed three times with fresh medium in order to remove NPs and incubated again at 37 1C until 48 h. Finally, after transfection, luciferase activity was measured as RLU per mg protein using the luciferase assay system (Luciferase Assay System with Reporter Lysis Buffer, Promega) and BCA reagent (Sigma, UK).
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!