Immunoblotting was used to quantify the protein levels in peritoneal macrophages and cardiac fibroblast. The samples were resolved on criterion XT bis-tris 4–12% 18 well (Bio-Rad Inc. Hercules, California) gel and MOPs Buffer (Bio-Rad, Hercules, California). The kaleidoscope precision plus standard (Bio-Rad) was used to determine the molecular weight of the protein for immunoblotting, 10–15 μg of protein lysate per sample was denatured and resolved by using criterion XT bis-tris 4–12% 18-gel (Bio-Rad I, Hercules, California.) gel in MOPs Buffer (Bio-Rad)., then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, California), and blocked with 5% nonfat milk. The membrane was probed with COX-2 (1:1000) and 5LOX (1:200) overnight at 4˚C, followed by secondary antibody (Bio-Rad, Hercules, California). The proteins were detected using the Femto chemiluminescence detection system (Pierce Chemical, Rockford, IL). Densitometry was performed using Image J software (NIH, USA).
Mops buffer
Manufactured by Bio-Rad
Sourced in United States, France
MOPS buffer is a common buffer solution used in various biochemical and molecular biology applications. It is a zwitterionic buffer that maintains a stable pH range, typically around 6.5 to 7.9. The buffer helps to maintain the desired pH environment for experiments involving proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Fluorchemfc2 device, by Cell Biosciences (2 mentions)
Alphaview software, by Cell Biosciences (2 mentions)
Femto chemiluminescence detection system, by Thermo Fisher Scientific (2 mentions)
Nonfat milk powder, by Bio-Rad (2 mentions)
Proteinase inhibitor, by Thermo Fisher Scientific (1 mentions)
Goat anti actin hrp, by Santa Cruz Biotechnology (1 mentions)
Criterion xt gel, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
Sds sample buffer, by Bio-Rad (1 mentions)
Phosphorimager screen, by GE Healthcare (1 mentions)
Imagequant tl v2003, by GE Healthcare (1 mentions)
Typhoon 9400, by GE Healthcare (1 mentions)
Ab9484, by Abcam (1 mentions)
Criterion xt, by Bio-Rad (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Cox 2, by Abcam (1 mentions)
5 lox, by Abcam (1 mentions)
9 protocols using mops buffer
1
Quantifying Protein Levels in Macrophages and Fibroblasts
2
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LV infarct tissues were weighted and placed in their respective 1.5 mL tubes with 16 μl of 1xPBS (without calcium, Invitrogen) and 1x proteinase inhibitor (Roche Diagnostics). While at 4°C, the samples were dis-membrated in short, 10 second intervals using a sonic dismembrator until homogenous, then centrifuged at maximum speed (14,000 rpm). The supernatant was used as the soluble protein fraction. The kidney protein was extracted using RIPA buffer. Total protein determined with Bradford assay. Electrophoresis of 10 μg of protein from each tissue with XT bis-tris-4-12% gel (Bio-Rad Inc.) in MOPS buffer (Bio-Rad) was performed. Samples were then transferred onto nitrocellulose membrane (Bio-Rad Inc.) and a total protein stain was performed with Pierce reversible protein stain, nitrocellulose membrane kit (Thermo Scientific Inc.). Each membrane was blocked for 1 hour at room temperature with 5% non-fat milk powder (Bio-Rad) dissolved in PBS-T and probed with primary antibodies. Primary antibodies (FPR2/ALX 1:1000; Ccl2 1:5000; arg-1 1:5000, GPR40 1:1000; 5LOX 1:1000) were probed overnight at 4°C, subsequently washed with PBS-T, and a secondary antibody was applied (Biorad). Proteins were detected using Femto chemiluminescence detection system. Densitometry of protein blots were assessed with ImageJ software (NIH, USA).
3
Western Blot Analysis of Rat Colonocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated rat colonocytes were lysed in RIPA buffer containing a protease inhibitors cocktail (Roche). Total protein extracts (30 μg) were loaded into 4–12% Criterion XT gel (Bio-Rad) before electrophoresis in MOPS buffer (Bio-Rad). After transfer on nitrocellulose membrane and incubation in blocking solution (TBS pH 7.5, 0.05% Tween 20 and 5% (weight:volume) BSA, membranes were incubated overnight (4 °C) with a primary antibody directed against activated-caspase 3 (Abcam 2303, rabbit, 1/1000) or proliferating cell nuclear antigen (PCNA, Abcam 29, mouse, 1/1000) or claudin-1 (Invitrogen, 717800, rabbit, 1/250) diluted in the blocking solution. After washes, blots were incubated for 2 h at room temperature with an anti-rabbit or anti-mouse HRP-linked secondary antibody (Jackson Immuno Research Laboratories, 1/5000) or a goat anti-actin-HRP (Santacruz Biotechnologies C-11, 1/1000) diluted in the blocking solution. After 3 washes, detection was performed by chemiluminescence using Clarity Western ECL substrate (Biorad) and the FluorChemFC2 device with AlphaView software (Cell Biosciences).
4
SDS-PAGE analysis of 35S-labeled macromolecules
5
Western Blot Analysis of Tight Junction Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen colonic tissue was homogenized in a lysis buffer [15 (link)], and 25 µg of total protein lysates were loaded onto 4%–12% Criterion XT gel (Bio-Rad, Marnes-La-Coquette, France) before electrophoresis in MOPS buffer (Bio-Rad, Marnes-La-Coquette, France). After transfer onto nitrocellulose membrane and incubation in blocking solution (TBS pH 7.5, 0.05% Tween 20, and 5% (wt/vol) non-fat dry milk), membranes were incubated overnight (4 °C) with rabbit ZO-1 antibody (1/250, 617300, Invitrogen, Cergy Pontoise, France) or with mouse claudin-1 antibody (1/250, 374900, Thermo-Fisher Scientific, Bedford, MA, USA) diluted in blocking solution. After three washes, blots were incubated for 2 h at room temperature with an anti-rabbit or anti-mouse HRP-linked secondary antibody. Revealing was performed using enhanced chemiluminescence (ECL system, Pierce Biotechnology, Courtabœuf, France), and bands were quantified by densitometry using the FluorChem FC2 device and the AlphaView software (Cell Biosciences, Santa Clara, CA, USA). GAPDH expression (Ab9484, mouse monoclonal antibody, Abcam, Cambridge, UK) was used to ensure consistent protein loading and transferring.
6
Western Blot Analysis of Tagged PfVAP1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the detection of tagged PfVAP1, IE were collected and saponin-lysed. Parasites were washed three times in PBS containing protease inhibitors and the pellet resuspended to obtain 106 IE μl−1. Samples were run on 10% Tris-Bis gels (BioRad) in MOPS Buffer (BioRad) and transferred onto nitrocellulose membranes (iBlot, Life Technologies). Membranes were blocked in 5% milk/0.05% tween 20 (Promega) in PBS (PBS/milk/tween) for 30 min at room temperature and incubated overnight with either mouse anti-Ty1 (1:100) or rabbit anti-HA (1:250). Following three washes with PBS/milk/tween, alkaline phosphate-conjugated secondary antibodies were added for 1 h at room temperature.
7
Western Blot Analysis of Inflammatory Mediators
8
Fab Fragment Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fab (3 μg/lane) was loaded on
a 4%–12% Bis-Tris precast gel (Biorad) in nonreducing conditions
and run at 120 V in a 3-morpholinopropane-1-sulfonic acid (MOPS) buffer
(Biorad). Bands were visualized with Imperial Protein Stain (Thermo
Fisher Scientific), and the size of the fragments was evaluated by
running a protein standard ladder (Biorad). The Fab bands were cut
and reduced by 10 mM TCEP at 37 °C and then alkylated in 40 mM
IAA at RT in the dark, followed by alkylation in 40 mM IAA at RT in
the dark. The Fab bands were digested by trypsin, chymotrypsin, thermolysin,
and alpha lytic protease at 37 °C overnight in a 50 mM ammonium
bicarbonate buffer. The peptides were extracted with two steps of
incubation at RT in 50% ACN and 0.01% TFA, and then 100% ACN, respectively.
The peptides were dried in speed-vac. To obtain the sequence of the
glycosylated Fab, the N-linked glycan was removed by PNGaseF at 37
°C overnight and then in gel digested as described above.
a 4%–12% Bis-Tris precast gel (Biorad) in nonreducing conditions
and run at 120 V in a 3-morpholinopropane-1-sulfonic acid (MOPS) buffer
(Biorad). Bands were visualized with Imperial Protein Stain (Thermo
Fisher Scientific), and the size of the fragments was evaluated by
running a protein standard ladder (Biorad). The Fab bands were cut
and reduced by 10 mM TCEP at 37 °C and then alkylated in 40 mM
IAA at RT in the dark, followed by alkylation in 40 mM IAA at RT in
the dark. The Fab bands were digested by trypsin, chymotrypsin, thermolysin,
and alpha lytic protease at 37 °C overnight in a 50 mM ammonium
bicarbonate buffer. The peptides were extracted with two steps of
incubation at RT in 50% ACN and 0.01% TFA, and then 100% ACN, respectively.
The peptides were dried in speed-vac. To obtain the sequence of the
glycosylated Fab, the N-linked glycan was removed by PNGaseF at 37
°C overnight and then in gel digested as described above.
9
Quantitative Immunoblot Analysis of Mitochondrial Proteins
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!