Cells were washed with cold PBS and lysed in high-salt lysis buffer (50 mM HEPES, 500 mM NaCl, 1.5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1% Triton X-100, pH 7.5) supplemented with 1 mM Na3VO4 (Sigma) and 1 mM complete ULTRA tablets mini EDTA-free Easy pack (Roche). Protein concentration was determined using the DC Protein Assay (Bio-Rad) with bovine serum albumin as a standard. Immunoblotting and zymography were then conducted as previously described [38 (link)]. Antibodies can be found in Additional file 1 : Table S1. Blots were imaged on a ChemiDoc MP (Bio-Rad) after incubating in ProSignal Pico ECL Spray (Genesee Scientific) for 3 min. Quantification was performed using ImageJ.
Complete ultra tablets mini edta free easy pack
Manufactured by Roche
Sourced in Switzerland, Germany
The Complete ULTRA tablets mini EDTA-free Easy pack is a laboratory equipment product designed for sample preparation. It provides a convenient and efficient way to handle small sample volumes. The product's core function is to facilitate the collection and processing of biological samples in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop easypack, by Roche (5 mentions)
Na3vo4, by Merck Group (4 mentions)
Dc protein assay, by Bio-Rad (4 mentions)
Prosignal pico ecl spray, by Genesee Scientific (3 mentions)
Chemidoc mp, by Bio-Rad (3 mentions)
Laemmli buffer, by Bio-Rad (3 mentions)
Trans blot turbo system, by Bio-Rad (3 mentions)
Synthetic melanin, by Merck Group (2 mentions)
Spectrafluor, by Tecan (2 mentions)
Ripa buffer with triton, by Bioworld Technology (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chemidoctr xrs, by Bio-Rad (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Image lab version 3, by Bio-Rad (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Standard dc protein assay, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
15 protocols using complete ultra tablets mini edta free easy pack
1
Immunoblotting and Zymography with Cell Lysis
2
Isolation of Mouse Brain Microvessels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary mouse brain microvessels were isolated from WT and claudin-12lacZ/lacZ C57BL/6J mice as previously described in detail [33 (link)]. The only modification to this previous protocol was that instead of the final plating step, microvessels were incubated in a red blood cell lysis buffer (0.83% ammonium chloride and Tris–HCl, pH = 7.5), for 5 min, RT. After two washing steps, microvessels were lysed in HES lysis buffer (10 mM HEPES, 1 mM EDTA solution, 250 mM sucrose solution), in the presence of protease inhibitor cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack (1 tablet/10 mL) (Roche Diagnostics, Mannheim, Germany), and kept at − 20 °C.
3
Protein Expression and Western Blotting Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were obtained using RIPA buffer/Triton pH 7.4 (Bioworld) with inhibitors of phosphatases (PhosSTOP EASYpack, Roche) and proteases (cOmplete ULTRA tablets Mini EDTA-free EASYpack, Roche), following commercial brand indications. Protein concentration was determined by BCA Protein Assay Kit (Thermo Scientific Pierce). Subsequently, extracts were diluted in Laemmli buffer (Bio-Rad), heated for 5 min at 98 °C and electrophoresis was performed in acrylamide/bisacrylamide gels in denaturing conditions (SDS-PAGE), using a Miniprotean base, and Western Blotting by Transblot Turbo system (Bio-Rad) to mmobilon-P PVDF membrane (Bio-Rad). Membranes were incubated with blocking solution of skimmed milk 5% in TBS with 0.1% Tween20, for at least one hour at room temperature shaking. Afterwards, the membranes were incubated with primary antibodies overnight at 4 °C shaking (Suppl. Table 1 ), washed with TBS-T and incubated with a secondary antibody conjugated with peroxidase (Suppl. Table 1 ) for 2 h at room temperature. Quimioluminiscence revealing (kit ECL Plus, Amersham) was carried out using the high resolution system ChemiDocTR XRS+ (Bio-Rad). Bands were digitalized using Image Lab version 3.0.1 (Bio-Rad) software.
4
Quantifying Intracellular and Extracellular Melanin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to measure hyperpigmentation, the protocol described by Bellei et al., 2008 [22 (link)] was followed, with some modifications. Intracellular and extracellular melanin were measured. Cell culture medium was collected in order to measure extracellular melanin. For intracellular melanin content measurement, cells were first washed with cold PBS and lysed with RIPA buffer with Triton, pH 7.4 (Bioworld), containing protease (complete ULTRA tablets Mini EDTA-free EASYpack, Roche) and phosphatase (PhosSTOP EASYpack, Roche) inhibitors. Lysates were then centrifuged at 17,000× g for 10 min at 4 °C. Supernatants were used to measure protein concentrations. The intracellular melanin pigments present in the pellets and the extracellular melanin pigments were solubilized by incubation in 1 M NaOH for 2 h at 60 °C. Absorbance was measured at 405 nm using a plate reader (SpectraFluor, Tecan, Männedorf, Switzerland. For each experiment standard curves were prepared using synthetic melanin (0–250 μg/mL, Sigma). Data were normalized with respect to the protein content and the non-irradiated control.
5
Protein Extraction and Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with cold PBS and lysed in high salt buffer (50mM HEPES, 500mM NaCl, 1.5mM MgCl2, 1mM EDTA, 10% glycerol, 1% Triton X-100, pH 7.5) supplemented with 1mM Na3VO4 (Sigma) and 1mM complete ULTRA tablets mini EDTA-free Easy pack (Roche). Protein concentration was determined using the DC Protein Assay (Bio-Rad) with BSA as a standard. Immunoblotting and zymography were then conducted as previously described39 (link). Antibodies can be found in supplemental table 2 . Blots were imaged on a ChemiDoc MP (Bio-Rad) after incubating in ProSignal Pico ECL Spray (Genesee Scientific) for 3 minutes.
6
Western Blot Protein Quantification
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) with a protease inhibitor cocktail (cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack, Roche, Basel, Switzerland), which contains serine, cysteine, and aspartic protease inhibitors, and the Phosphokinase Inhibitor Cocktail Set V (Merck KGaA, Darmstadt, Germany) containing the serine and cysteine protease inhibitors, 4- benzenesulfonyl fluoride hydrochloride, Aprotinin, E-64, and leupeptin hemisulfate. Protein concentrations were measured using a standard DC protein assay (Bio-Rad, Hercules, CA, USA), and 40 μg of protein was separated by SDS-PAGE and transferred to a PVDF membrane. After being blocked with 5% BSA for 1 h at room temperature, the membrane was incubated with primary antibodies diluted at 1:1000 overnight at 4°C, washed three times with TBST and incubated with secondary antibody for 1 h at room temperature. After being washed three times with TBST, the membranes were developed with New Clarity™ Western ECL Substrate (Bio-Rad), imaged and analyzed with a ChemiDoc™ XRS system and Quantity One software (Bio-Rad).
7
Protein Extraction and Analysis Protocol
8
Melanin Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hyperpigmentation was measured following the protocol described by Bellei et al., 2008 [36 (link)], with some modifications. Cell culture medium was collected for extracellular melanin pigments measurement. Cells were washed with cold PBS and lysed with RIPA buffer with Triton, pH 7.4 (Bioworld), containing protease (complete ULTRA tablets Mini EDTA-free EASYpack, Roche) and phosphatase (PhosSTOP EASYpack, Roche) inhibitors. Lysates were then centrifuged at 17,000 g for 10 min at 4°C. Supernatants were used for protein concentration measurements. Pellets contained the intracellular melanin pigments, which were solubilized by incubation in 1 M NaOH for 2 h at 60°C. Extracellular melanin pigments were also solubilized, and absorbance was measured at 405 nm using a plate reader (SpectraFluor, Tecan). Standard curves were prepared for each experiment using synthetic melanin (0-250 μg/mL, Sigma). Data were normalized with respect to the protein content and then to the non-irradiated control.
9
Western Blot Analysis of p53 and β-catenin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular extracts were obtained with RIPA buffer with Triton, pH 7.4 (Bioworld), containing phosphatase (PhosSTOP EASYpack, Roche) and protease (complete ULTRA tablets Mini EDTA-free EASYpack, Roche) inhibitors, following the manufacturer’s instructions. Protein concentration was determined using the BCA Protein Assay Kit (Pierce). Cellular extracts were diluted in Laemmli buffer (Bio-Rad) and heated for 5 min at 98°C. Electrophoresis was performed using acrylamide/bisacrylamide gels in denaturing conditions (SDS-PAGE) using a Mini-PROTEAN cell. Western blotting onto PVDF membranes (Bio-Rad) was performed using a Transblot Turbo system (Bio-Rad). Membranes were incubated with a after blocking solution consisting of skimmed milk in 0.1% TBS-Tween 20, with primary antibodies (anti-p53 and anti-β-catenin), and peroxidase-conjugated secondary antibodies (Thermo Fisher), following the manufacturer’s instructions. Protein bands were visualized by chemiluminiscence (ECL Pl us Kit, Amersham) using the high resolution ChemiDocTR XRS+ system (Bio-Rad), and digitalized using Image Lab version 3.0.1 software (Bio-Rad).
10
Immunoprecipitation and Immunoblotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All steps were conducted on ice or at 4 °C. All beads were washed three times with five volumes TBS before use. Cells were lysed in RIPA buffer containing 1 mM Na3VO4 (Sigma) and 1 mM complete ULTRA tablets mini EDTA-free Easy pack (Roche) and agitated for 30 min prior to centrifugation at 10,000×g for 10 min. Protein concentrations were determined via DC protein assay (Bio-Rad), and 100 μg of protein was added to IgG control beads (Cell Signaling, 5873S or 8726S) or 6 μg of the indicated antibody before incubating overnight. Magnetic beads (Active Motif, 53,033) were then added to the antibody/protein mixture and allowed to incubate for an additional 4 h. Tubes were then placed on a magnetic separator, and beads were washed three times with TBS before being resuspended and boiled for 5 min in 2× Laemmli sample buffer lacking reducing agent. β-mercaptoethanol was then added, and samples were again boiled for 5 min before immunoblotting.
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!