HAP1 parental fibroblasts were seeded into 24-well culture plates (CytoOne CC7682–7524) and grown to 75% confluence. Cells were then trypsinized, followed by recovery in growth medium on an orbital shaker for 10 min. Cells were pelleted at 200xg, washed once in PBS, and then lysed using Cell Lytic M reagent (Sigma C2978) containing 1X protease inhibitor cocktail (Sigma P8340) for 45 min on ice. Cell lysates were centrifuged for 10 min at 15000 rpm at 4C. The supernatant (soluble fraction) was diluted in LDS Sample buffer (Life Technologies #NP0007) and Reducing buffer (Life Technologies #NP00009). The pellet (insoluble fraction) was the resuspended in LDS Sample buffer and Reducing buffer. The two fractions were boiled at 95C for 5 min and subsequently analyzed by western blotting. Note that the actual fractions obtained by this method may vary from previous reports (Schiffhauer et al., 2019 (link)) since we did not use ATP in our lysis buffer, which would promote release of MII from the insoluble fraction into the soluble fraction. Therefore, our values for the insoluble fraction tend to be higher.
Reducing buffer
Manufactured by Thermo Fisher Scientific
Sourced in Canada
Reducing buffer is a laboratory reagent used to maintain a reducing environment in protein samples. It contains reducing agents that help maintain the native structure and activity of proteins by preventing oxidation.
Automatically generated - may contain errors
Lab products found in correlation
Cell lysis buffer, by Cell Signaling Technology (3 mentions)
Complete ultra protease inhibitor cocktail, by Roche (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (3 mentions)
Mini protean tgx gel, by Bio-Rad (3 mentions)
Lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Sample buffer, by Thermo Fisher Scientific (2 mentions)
Mouse ho 1 elisa set, by Enzo Life Sciences (2 mentions)
Precellys evolution, by Bertin Technologies (2 mentions)
Lithium dodecyl sulfate (lds), by Thermo Fisher Scientific (2 mentions)
Phosstop, by Merck Group (2 mentions)
Dounce tissue grinder set, by Merck Group (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Cell lytic m reagent, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bicinchoninic acid protein assay, by Merck Group (1 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Nupage loading dye, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
12 protocols using reducing buffer
1
Subcellular Fractionation and Western Blotting
2
Western Blot Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Different samples for western blot (including cells, human retinal explants or mice retinas) were lysed in RIPA buffer with a 1% protease/phosphatase inhibitor cocktail (Cell Signalling Technology, Danvers, MA, 5872S), centrifuged at 12,000× g at 4 °C for 10 m and the supernatant was collected. Protein concentration measurement was conducted with a bicinchoninic acid protein assay (Sigma‐Aldrich, Burlington, MA, BCA1-1KT). Cell lysates with the same amount were heated for 10 m at 70 °C, adding NuPAGE loading dye and reducing buffer (Life Technologies, Carlsbad, CA). After loading onto NuPAGE 4-12% Bis-Tris Protein gels (Thermo Fisher Scientific, Waltham, MA), the samples were electrophoresed at 180 V, 4 °C for 70 m. The proteins were transferred to PVDF membranes (Millipore, Burlington, MA) using the iBlot gel transfer device (Thermo Fisher Scientific, Waltham, MA). The PVDF membranes were blocked with 5% bovine serum albumin, Tris-Buffered Saline and 0.1% Tween 20 for 1 hr at room temperature and then incubated with primary antibody overnight at 4°C and secondary antibody for 2 h at room temperature. Antibodies used, including their company, species, catalogue number and working dilution, are listed in Table 2 . Protein expression was visualised using ECL (Bio‐rad, Hercules, CA, 170-5061) and quantified with the Gene Tools image scanning and analysis package.
3
Anatomical Sampling of Arcuate Nucleus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immediately after euthanasia, the brains were immediately removed and frozen at −80°C. Sections of the forebrain (500 μm) were cut in a cryostat. The region of the ARC was identified (approximately 6.5 mm rostral to the interaural line; Paxinos and Watson, 1986 ) in the sections and using a circular 1 mm (internal diameter) micro-punch tool, 500 μm thick punch-outs of the ARC, bilaterally, were taken and immediately homogenized in cold radioimmunoprecipitation assay buffer (50 mM Tris, 150 mM NaCl, 1% Triton-X 100, 0.25% sodium deoxycholate, 1 mM NaF, 1 mM sodium orthovanadate, 25 mM β-glycerophosphate) with protease inhibitor cocktail (Roche Applied Science; Laval, QC) by an electric homogenizer (VWR International; Radnor, PA). Homogenates were then sonicated over three passages for 15 s each on ice (55%; Sonic Dimembrator Model 150; Fisher Scientific). Samples were then rotated for 10 min at 4°C and centrifuged at 4°C for 20 min at 14,000 RPM. Protein content of homogenates was quantified using the Bio-Rad Dc protein assay kit (Bio-Rad Laboratories; Hercules, CA). Protein samples were added to 25% sample buffer and 10% reducing buffer (Life Technologies; Burlington, ON) and water to a standard protein concentration of 1.67 mg/ml (Messenger and Ciriello, 2013 (link); Moreau and Ciriello, 2013 ).
4
Western Blot Analysis of Lipid-Associated Proteins
5
SDS-PAGE Protein Separation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five to 10 μg of each LC protein was added to 7.5 μL LDS sample loading buffer (4x; Invitrogen, Carlsbad, CA), and for reduced samples, 2.5 μL reducing buffer (10x; Invitrogen) was also added. All samples were boiled 10 min prior to loading onto 4–12% Bis-Tris SDS-PAGE gels (Invitrogen). Electrophoresis was performed using a Novex apparatus (ThermoFisher Scientific, Waltham, MA) with MES buffer at 200 V for 30 min. Gels were stained using 1% Coomassie (1% Brilliant blue R250, 50% methanol, 10% acetic acid) for 5 min followed by 15 min incubation in destaining solution (50% methanol/10% acetic acid) and then a water incubation for up to 24 hr.
6
Immunoprecipitation of HA-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neurons and HEK-293 cells were grown, transfected and lysed following the above protocol, except IP Buffer was used to lyse cells (10% glycerol, 1% NP-40, 50 mM Tris, 200 mM NaCl, 2 mM MgCl2, cOmplete Mini, and PhosStop). The lysates were thawed and loaded (250 µg for neurons and 500 µg for HEK-293 cells) onto Pierce Anti-HA magnetic beads (Thermo Fisher Scientific), and immunoprecipitated following the manufacturer’s protocol. After the final wash, the beads were resuspended in 25 µl of 1× sample buffer (Invitrogen) and reducing buffer (Invitrogen), and then boiled for 5 min at 95°C. After boiling, everything (excluding the beads) was loaded onto the SDS–PAGE gel. Lysate lanes were loaded using 5% of total protein used for the IPs.
7
Western Blot Analysis of Gpx4 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDMs were lysed with cell lysis buffer (Cell Signaling) supplemented with 2 mM PMSF (Sigma-Aldrich) and a complete Ultra protease inhibitor cocktail (Roche). Protein concentrations were determined by using Pierce 660-nm Protein Assay Reagent (Thermo Fisher Scientific) and normalized, and the protein samples were denatured by adding reducing buffer (Thermo Fisher Scientific) before incubation at 95°C for 5 min. Samples were separated in Mini-Protean TGX gels (Bio-Rad) and then transferred to the nitrocellulose membrane by using Trans-Blot Turbo Transfer System Bio-Rad machine according to manufacturer’s instructions. Nitrocellulose membranes were blocked by using Odyssey blocking buffer (LI-COR) at room temperature for 2 h. Immunoblotting was performed by adding either anti-Gpx4 (1:1,000, clone EPNCIR144; Abcam) or anti-β actin (1:1,000, clone 8H10D10; Cell Signaling) and the membranes incubated at 4°C overnight. Subsequently, membranes were washed with 0.02% Tween20 (Sigma-Aldrich) in 1× DPBS and then treated with the respective secondary detection reagent (IRDye 800CW Donkey anti–rabbit IgG [LI-COR] or IRDye 680CW Donkey anti–mouse IgG [1:10,000; LI-COR]) at room temperature for 1 h. After washes in 0.02% DPBS-Tween20, membranes were imaged on a LI-COR Odyssey.
8
Quantifying Protein Expression in Mtb-Infected Lungs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Perfused lungs from naïve and Mtb-infected mice were disrupted in 2 ml tubes containing 2.7 mm glass beads and PBS with cOmplete ULTRA™ protease inhibitor cocktail (Roche, Basel, Switzerland) and 2mM of PMSF (Sigma-Aldrich, Saint Louis, MO), using a Precellys Evolution™ tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). Cell cultures were lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA) containing cOmplete ULTRA™ protease inhibitor cocktail (Roche, Basel Switzerland) and 2mM of PMSF (Sigma-Aldrich, Saint Louis, MO). For western blot, samples were denatured for 5 min at 95°C in reducing buffer (ThermoFisher Scientific, Waltham, MA), separated in mini-protean TGX gels (Bio-Rad, Hercules, CA) and transferred to PVDF membranes prior to staining with specific antibodies and development with luminol. Protein expression in samples was calculated utilizing ImageJ software in relation to β-actin expression. The concentration of HO-1 in samples was measured by ELISA using mouse HO-1 ELISA set (Enzo Life Sciences, Farmingdale, NY). Antibody specifications are provided in Table S1 .
9
Quantifying Protein Expression in Mtb-Infected Lungs
10
ASC Oligomerization Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Specks were incubated with the indicated dose of VHHASC, mutVHHASC or anti‐ASC pAb for 1 h at RT. DSS was added to a final concentration of 2 mM and further incubated at RT. Cross‐linking reaction was halted by mixing with electrophoresis samples buffer (either LDS‐sample buffer complemented with reducing buffer – ThermoFisher Scientific; or provided for WES readout by ProteinSimple) and heating at 95°C for 5 min. Samples were then run on WES following manufacturer's instruction or run for Western blot on a 4–12% SDS‐PAGE (ThermoFisher Scientific – using MOPS buffer, ThermoFisher Scientific) and transferred onto a PVDF membrane (Immobilon FL, Merck‐Millipore) prior to blocking with 3% BSA (w/v) in TBS buffer and detection of ASC distribution (monomeric to oligomeric state) using a monoclonal anti‐ASC antibody (N‐15).
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!