GST-tagged proteins from pBG101 vectors were purified from ArcticExpress Escherichia coli (Agilent Technologies). Bacterial pellets were resuspended in NET buffer [25 mM tris (pH 8), 50 mM NaCl, 0.1 mM EDTA, 5% glycerol, 1× Roche cOmplete Mini EDTA-free protease inhibitor, and 1 mM DTT] and sonicated. Triton X-100 was added to a final concentration of 1%, and lysates were incubated on ice for 30 min. Following centrifugation, the cleared supernatant was incubated with glutathione-Sepharose beads (GE Healthcare, catalog no. 17-0756-01) for 2 hours at 4°C. Beads were washed three times with NET buffer containing 1% Triton X-100, and bound proteins were recovered with elution buffer [10 mM Hepes (pH 7.5), 15 mM glutathione, 1× Roche cOmplete Mini EDTA-free protease inhibitor, and 1 mM DTT]. Purified proteins were dialyzed into 10 mM Hepes (pH 7.5), 50 mM NaCl, and 1× Roche cOmplete Mini EDTA-free protease inhibitor.
Complete mini edta free protease inhibitor
Manufactured by Roche
Sourced in Switzerland, Germany, United States, United Kingdom
The Complete Mini EDTA-free protease inhibitor is a laboratory product designed to inhibit proteases in protein samples. It contains a mixture of inhibitors that target a broad range of proteases without the use of EDTA.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Odyssey infrared imaging system, by LI COR (6 mentions)
Phosstop easypack, by Roche (5 mentions)
Phosstop phosphatase inhibitor, by Roche (3 mentions)
Phosphatase inhibitor cocktail 2 and 3, by Merck Group (3 mentions)
Phosstop, by Roche (3 mentions)
Phenylmethene sulfonylfluoride pmsf 10 mm, by Merck Group (3 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Glutathione sepharose bead, by GE Healthcare (2 mentions)
X cell 2 mini electrophoresis apparatus, by Thermo Fisher Scientific (2 mentions)
Ir dye 800 cw and ir dye 680 secondary antibodies, by LI COR (2 mentions)
Ultrapure water, by Thermo Fisher Scientific (2 mentions)
Strataclean resin, by Agilent Technologies (2 mentions)
Ab1791, by Abcam (2 mentions)
Celltrics filter, by Sysmex (2 mentions)
Ht fusion, by PerkinElmer (2 mentions)
Pathscan phospho s6 ribosomal protein ser235 236 sandwich elisa kit, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Image studio lite, by LI COR (2 mentions)
92 protocols using complete mini edta free protease inhibitor
1
Purification of GST-tagged Proteins
2
Proteomic Profiling of BRD4 Interactome
3
Western Blot Analysis of Immune Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were collected in 1×RIPA buffer (Cell Signaling Technology, Inc., Danvers, MA) supplemented with Complete Mini, EDTA-free protease inhibitor (Roche Applied Science, Indianapolis, IN). Protein concentration was assayed by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Proteins were resolved on 4–20% SDS-PAGE gel and transferred to PVDF membrane using Mini Trans-Blot Electrophoretic Transfer Cell apparatus (Bio-Rad, Hercules, CA). Primary antibodies used for western blot analysis were rabbit monoclonal RIG-I (D14G6), cleaved CASP3 (Asp175) (5A1E), cleaved PARP (Asp214) (D64E10) XP (Cell Signaling technology, Inc., Danvers, MA), IRF3 antibody [EP2419Y] (Abcam, Cambridge, MA), and goat polyclonal anti-actin (I-19) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Secondary antibodies used were HRP-linked goat anti-rabbit IgG (Cell Signaling technology, Inc., Danvers, MA) and HRP-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immune complexes were visualized with Amersham ECL Western Blotting Detection Reagents and exposed to X-ray films (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) according to the manufacturer’s instruction. The protein level was quantitated by densitometry measurement using AlphaEaseFC software (Alpha Innotech, Miami, FL).
4
Western Blotting and Cytokine Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer containing Complete Mini EDTA-free protease inhibitor (Roche) and PhosSTOP phosphatase inhibitor (Roche, Mississauga, ON, Canada). Total protein was quantified and equal concentrations were submitted to SDS-PAGE by standard methods. Proteins were transferred to PVDF membrane and probed with antibodies raised against GFAP (Sigma, St. Louis, MO, USA), GS (Abcam, Cambridge, MA, USA), phospho-p38 and pan-p38 MAPK (Cell Signaling, Danvers, MA, USA), IκBα (Santa Cruz, Dallas, TX, USA), TNF-α (R&D Systems, Minneapolis, MN, USA), and GAPDH (Calbiochem, San Diego, CA, USA), and detected with appropriate IRDye secondary antibody (Li-Cor Biosciences, Lincoln, NE, USA). Blots were imaged with an Odyssey infrared imaging system (Li-Cor Biosciences). For TNF-α and multiplex cytokine profiling, conditioned media was snap frozen and submitted for laser bead analyses on a Bioplex 200 to detect sensitive and quantitative target protein concentrations against a standard curve (Eve Technologies).
5
Cmr2 Co-Immunoprecipitation and RNA Cleavage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultures were grown to late log phase, harvested by centrifugation, and weighed. Lysis buffer (50 mM Tris-Cl at pH 8, 30 U/mL SUPERase• In [Life Technologies], 1× Complete-mini EDTA-free protease inhibitor [Roche]) was added (3 mL per 1 g of cells). Cells were lysed by sonication and centrifuged at 20,000g for 30 min at 4°C. The soluble fraction was collected as S20 cell extract, with protein quantified by Qubit assay (Life Technologies).
IgY antibodies previously raised against recombinant Cmr2 (Hale et al. 2012 (link)) were used for co-IPs. Co-IPs were carried out as previously described (Hale et al. 2012 (link)) with the following modifications: Each immunoprecipitation (30 µL of resin) used S20 cell extract containing 2 mg of protein. UltraLink hydrazide (Pierce) resin was used for silver stain analysis of immunoprecipitations, and anti-IgY agarose resin (Gallus Immunotech) was used for RNA cleavage assays.
IgY antibodies previously raised against recombinant Cmr2 (Hale et al. 2012 (link)) were used for co-IPs. Co-IPs were carried out as previously described (Hale et al. 2012 (link)) with the following modifications: Each immunoprecipitation (30 µL of resin) used S20 cell extract containing 2 mg of protein. UltraLink hydrazide (Pierce) resin was used for silver stain analysis of immunoprecipitations, and anti-IgY agarose resin (Gallus Immunotech) was used for RNA cleavage assays.
6
Protein Lysis and Immunoblotting Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% v/v NP-40, 0.25% w/v Na-deoxycholate, 0.1% w/v SDS, 10 mM NaF, 2 mM Na₃VO₄ and 1 tablet/10 ml, Complete, Mini, EDTA-free protease inhibitor (Roche, Welwyn Garden city, U.K.)). Samples were incubated for 10mins on ice, centrifuged at 13 000 rpm, 10 min and 4°C, and supernatants (RIPA soluble protein lysates) were collected. Standard SDS–PAGE and immunoblotting protocols were performed with 20 µg of protein per lane loaded on SDS–PAGE gels. The following primary antibodies were used for immunoblotting: HIF-1α (610958, BD Biosciences (Workingham, U.K.)), HIF-1β (3718, CST ((Leiden, Holland)), HIF-2α (7096, CST ((Leiden, Holland)), Actin (60009-1, Proteintech (Manchester, U.K.)), BAF155 (11956, CST ((Leiden, Holland)), KDM5A (3876, CST ((Leiden, Holland)). Three biological replicates were analysed per condition. Immunoblot figures are from one biological replicate, which is representative of all replicates.
7
BALF Analysis for Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The airway lumina of the sacrificed animals were washed using a tracheal cannula with 1 ml of saline twice. The BALF so obtained, was centrifuged and supernatants were stored at −80°C and subjected to ELISA assay using kits. Total and differential cell numbers were counted double-blind, using a hemocytometer. Cytospin cell preparations were made by placing the cells onto glass slides, centrifuging at 4°C for 10 min at 1,000 × g, and staining with Diff-Quik. Lung tissues were homogenized in saline to a concentration of 100 mg/ml with the complete, Mini, EDTA-free Protease Inhibitor (Roche Applied Science) and the debris-free supernatant was used for cytokine measurement.
8
GFP-Fusion Protein Expression Analysis in A. baldaniorum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For analysis of the expression of GFP-fusion proteins in A. baldaniorum Sp245, derivative strains were cultured in NFB*+KNO3 medium at 30°C to an OD600 of 1.0. Bacteria cells were collected by centrifugation at 10,000 rpm for 5 min. The cells were resuspended in PBS, supplemented with Roche Complete Mini EDTA-free protease inhibitor, and ultrasonicated for 2 min. Cellular debris was sedimented by centrifugation (30 min. at 10,000 X g). The supernatant (soluble fraction) was obtained, and the pellet (insoluble fraction) was resuspended in PBS supplemented with detergent mix (0.5% Triton X 100; 0.1% SDS, and 0.5% 7-Deoxycholic acid sodium salt), and centrifuged at 25,000 X g for 2 h, to obtain protein membrane total extract. The total protein concentration of the respective fractions was determined using the Bradford method. The samples were resuspended in 5X loading buffer, boiled, separated on 10% SDS-PAGE, and subsequently transferred onto polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany) for immunoblotting using anti-GFP antibody (GFP D5.1 Rabbit mAb, HRP Conjugate, Cell Signaling Technology, Danvers, Massachusetts, USA.) HRP-DAB substrate Kit (Thermo Scientific Pierce) was used to check the target proteins on the membrane according to the manufacturer’s guidelines.
9
Protein Lysis and Immunoblotting in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated in serum-free medium plus 11 mM glucose ± agonist (100 nM) before lysis (20 mM Tris base, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100 plus complete mini EDTA-free protease inhibitor (Roche)), addition of urea sample buffer (100 mM Tris-HCl pH 6.8, 2.5% SDS, 4 M urea, 100 mM DTT, 0.05% bromophenol blue), 10 min incubation at 37 °C and immunoblotting.
10
FOLR1 Expression Analysis in Murine Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ovary, kidney and brain cortex were taken out from mice and lysed in a lysis buffer containing 7 ml Tris-EDTA buffer (10 mM Tris-HCl, 1mM EDTA), 1 tablet of Complete Mini EDTA-free protease inhibitor (Roche) and 1% (70 μl) IGEPAL®CA-630 (Sigma-Aldrich). 30 μg of proteins from ovary, kidney and cortex were used for Western blotting analysis. Primary FOLR1 antibody (see appendix 8) was applied in a concentration of 0.5 μg/ml in the blocking buffer and the membrane was incubated overnight at 4°C. The membrane was washed for 3 × 10 min in PBS-Tween and secondary HRP-conjugated polyclonal swine-anti-rabbit antibody (DAKO S3023) was applied in a dilution of 1:1,000 in blocking buffer and incubated for 1 h at RT. The membrane was incubated in a chemiluminescent detection reagent. [ECLTM Western Blotting Analysis system (GE Healthcare)] and developed for 45 s using a ImageQuant LAS-4000 (GE Healthcare).
Endoglycosidase H (New England Biolabs, P0702S) treatment was performed according to the manufactorers instructions (New England Biolabs protocol).
Endoglycosidase H (New England Biolabs, P0702S) treatment was performed according to the manufactorers instructions (New England Biolabs protocol).
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!