For in vitro pulldown, HEK293 grown in 100 mm dishes were single transfected with the desired plasmids. Twenty-four hours after transfection, cells were lysed in 1 ml lysis buffer (Miltenyi Biotec). For magnetic labeling, supernatants were incubated on ice with anti-tag MicroBeads of choice for 1 h. Cell lysates were applied onto equilibrated MicroColumns, washed four times with high salt buffer (500 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mM Tris-HCl at pH 8.0), and washed two times with wash buffer 2. Proteins were eluted by nondenaturing elution of the column-bound antigen by using a pH shift according to the manufacturer’s instructions. Small samples of the eluates were analyzed by LDS-PAGE and Western blot to check for purification. For CoIP, eluates were combined, filled up to 1 ml with lysis buffer (Miltenyi Biotec), and incubated overnight at 4°C. The next day, magnetic labeling, purification, and elution were done as described earlier. Eluted immunoprecipitates were analyzed by SDS-PAGE and Western blot.
Lysis buffer
Manufactured by Miltenyi Biotec
Sourced in Germany
Lysis buffer is a solution used to break down or 'lyse' cells, releasing the cellular contents, including proteins, nucleic acids, and other biomolecules. It serves as a core component in various biological assays and sample preparation protocols.
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Lab products found in correlation
Protease inhibitor cocktail, by Abcam (1 mentions)
M tube, by Miltenyi Biotec (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
μmacs ha isolation kit, by Miltenyi Biotec (1 mentions)
μ columns, by Miltenyi Biotec (1 mentions)
Glass dounce tissue grinder, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Mitochondria isolation kit, by Miltenyi Biotec (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Pepstatin a, by Research Products International (1 mentions)
Multimacs m96 separator, by Miltenyi Biotec (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Nu page lithium dodecyl sulfate sample buffer, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by AppliChem (1 mentions)
μmacs epitope tag protein isolation kit, by Miltenyi Biotec (1 mentions)
Gentle macs mechanical dissociator, by Miltenyi Biotec (1 mentions)
Percoll, by Merck Group (1 mentions)
Facsverse, by BD (1 mentions)
12 protocols using lysis buffer
1
In vitro Protein Pulldown Assay
2
Co-immunoprecipitation of zDHHC17 interactions
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For co-immunoprecipitation assays, HEK293T cells in 24-well plates were co-transfected with murine zDHHC17 in HA-pEF-BOS plasmid (or empty vector) and EGFP, EGFP-PAI-RBP1, or EGFP-SLAIN1 plasmids in triplicate wells. Following 24 h, cells in each well were lysed in 100 μl of lysis buffer (Miltenyi Biotech) and lysates from three identical wells were pooled for co-immunoprecipitation assays. Protein isolation was performed using μMACS HA Isolation kit (Miltenyi Biotec), according to the manufacturer's protocol; however, to preserve binding of proteins to HA-zDHHC17, all washes were performed in lysis buffer.
3
Protein-Protein Interactions via Co-immunoprecipitation
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Co-immunoprecipitation (CoIP) was performed by using μMACS epitope tag protein isolation kits (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to manufacturer's instructions. Briefly, 24 h after transfection, HEK293 cells were lysed in 1 ml lysis buffer (Miltenyi Biotec) containing 1x proteinase and phosphatase inhibitor (HALT, Thermo Fisher). Cellular debris were pelleted and 1 ml of supernatants were incubated with anti-tag MicroBeads for 1 h on ice. Afterwards, cell lysates were applied onto equilibrated μmacs columns and washed stringently to remove non-specific interacting molecules. Proteins were eluted with 1x pre-heated 95°C NuPage lithium dodecyl sulfate (LDS) sample buffer (Thermo Fisher) supplemented with 100 mM Dithiothreitol (DTT, AppliChem, Darmstadt, Germany). Raw lysates and eluates were analyzed by Western blot. For endogenous CoIP, PHNs were lysed in lysis buffer (Miltenyi Biotec) supplemented with 1x HALT. Lysates were centrifuged and supernatant was transferred to fresh Eppendorf cups. Lysate of PHNs or human tissue was adjusted to 1 ml with lysis buffer. Afterwards, lysates were incubated with 2 μg of the required antibodies for 2 h at 4°C. Afterwards, cell lysates were incubated with anti protein G MicroBeads (Miltenyi Biotec) for 1 h on ice. Immunoprecipitations were performed as described above.
4
Hippocampal Protein Extraction Protocol
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Mice were anesthetized using isoflurane before transcardially perfused with 1 × PBS. Mouse brains were removed and the hippocampus were dissected. The hippocampus tissues were washed by PBS and diced into 15–20 pieces before homogenization in M tubes (130-093-236, Miltenyi Biotec) with 100 mg/1 mL ice cold lysis buffer (Cat. No. ab156034, Abcam, Cambridge, UK) containing 1:100 protease inhibitor cocktail (Cat. No. P2714, Sigma-Aldrich, St. Louis, MO). M tubes were then transferred onto gentleMACS Dissociator (130-095-937, Miltenyi Biotec) for homogenizing using the gentleMACS program Protein_01.01. The homogenate was transferred to 1.5 mL Eppendorf and centrifuged at 12,000g for 20 min. The supernatants were collected, aliquoted and frozen at − 80 °C before use.
5
Isolating B-CTCs from Mouse Blood
6
Mitochondrial Isolation from Neurons
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Freshly harvested neurons (about 1 × 108 cells) were washed with PBS and resuspended in 1 mL ice-cold Lysis Buffer (Cat#130-096-946, Miltenyi Biotech, Auburn, CA, USA). After homogenization of the cells with a Dounce homogenizer, cell lysates were transferred to 15 mL conical tubes and mixed with 9 mL of ice-cold 1 × Separation Buffer with 50 µL Anti-TOM22 MicroBeads to magnetically label the mitochondria. The mixture was incubated for 1 h at 4 °C with gentle shaking. An LS Column for separation was placed in the magnetic field of a MACS Separator (MidiMACS Separation Unit) and rinsed with 3 mL of 1× Separation Buffer. The labeled cell lysates were applied onto the column stepwise (3 × 3.3 mL) and the lysate was allowed to run through. The column with the lysate was removed from the MACS Separator and placed on a 15 mL conical tube. An amount of 1× Separation Buffer (1.5 mL) was loaded on the column and immediately flushed out the magnetically labeled mitochondria by firmly pushing the plunger into the column. Isolated mitochondria were collected by centrifuging at 1000 rpm for 1 min.
7
Purification and Detection of HNV Proteins
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Cells transfected with HNV wt or mutant G expression plasmids were lysed in lysis buffer (Miltenyi Biotec). Cell lysates were incubated with 40ul of anti-HA microbeads at 4°C for 30 minutes with rotation. Lysates were purified and eluted over μ columns (Miltenyi Biotec). PAGE was used for cell lysates and column elutions using a 10% gel. F and G proteins were detected by Western blot analysis, as detailed above.
8
Isolation and Lysis of Mitochondria
9
Inflammatory and Barrier Marker Expression
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The expression of inflammation markers, like NF-κB, TNF-α, IL-1β and NLRP3, was analyzed. The mRNA expression of tight junction’s marker occludin and the glycocalyx marker galectin-3 was also investigated in the porcine model (Table 2 ). Briefly, after incubation, the corneas were cut into small pieces and incubated in 800 µL Lysis Buffer (Miltenyi Biotec, Köln, Germany) for 1 h at 37 °C on a shaker. The mRNA was isolated from porcine cornea explants and reverse transcribed using the MultiMACS mRNA and cDNA synthesis was done with cDNA Synthesis Kit on the MultiMACS™ M96 Separator (Miltenyi Biotec, Köln, Germany) according to the manufacturer’s protocol. After cDNA synthesis, a quantitative real-time PCR (qRT-PCR) was performed using the SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad, Feldkirchen, Germany) in a thermal cycler (Bio-Rad CFX96™ Real-Time System, Bio-Rad, Feldkirchen, Germany), as described previously [56 (link)]. β-actin (ACTB) and Ribosomal protein L 4 (RPL4) were used as housekeeping genes.
10
Transcriptional Profiling of p190-B-/- Cells
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LSK-SLAM were isolated from secondary recipients of WT or p190-B−/− cells. Cells were lysed in lysis buffer (Miltenyi Biotech) and samples were further processed at Miltenyi Biotech, Germany, using Agilent Whole Mouse Oligo Microarrays. Data were analysed by gene set enrichment analysis across the complete list of genes. Gene ontology analysis on top candidate genes based on Student's t-test analysis was performed using ToppGene Suite software. The accession number for the raw data is GSE89794.
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