Heparinized whole blood was incubated with or without GM-CSF (10ng/mL, 37°C, 15min) and red blood cells were lysed with lysing buffer (BD Pharmlyse™, BD Biosciences, San Jose, CA). After washing with ice-cold PBS, cells were extracted with protein extraction buffer (M-PER® #78501) containing protease inhibitor cocktail (0.5% v/v), phosphatase inhibitor cocktail (1% v/v) (all from Thermo Scientific, Rockford, IL), and EDTA (5 mM). Protein extracts were suspended in sample loading buffer, layered onto AnykD gradient gels (Bio-rad, Hercules, CA), separated by gel electrophoresis (100 V, 150 minutes) and transferred (Transblot™, Bio-rad, Hercules, CA) onto PVDF membranes (Immobilon, Merck Millipore, Billerica, MA), per manufacturer's instructions. Membranes were incubated with either murine anti-CD11b monoclonal antibody (10 μl from the vial diluted in 500 μl of immunoblotting buffer (Uchida 2009 (link)) (BioLegend, San Diego, CA) or murine anti-phospho- STAT5 antibody (5 μl from the vial diluted in 1000 μl of immunoblotting buffer (Millipore, Billerica, MA) and detection was done using ECL plus™ (GE healthcare, Little Chalfont, UK). Band intensity of CD11b was quantified by Image Quant TL Analysis Toolbox Ver 7.0 (GE Healthcare, Pittsburgh, PA).
Anykd gradient gel
Manufactured by Bio-Rad
Sourced in United States
AnyKD gradient gels are polyacrylamide gels designed for protein separation and analysis. They feature a continuous gradient of polyacrylamide concentration, allowing for the separation of a wide range of protein sizes in a single gel.
Automatically generated - may contain errors
Lab products found in correlation
Laemmli buffer, by LI COR (2 mentions)
Blocking buffer, by LI COR (2 mentions)
Odyssey infrared imager, by LI COR (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Irdye conjugated secondary antibody, by LI COR (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bradford reagent, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Immobilon, by Merck Group (1 mentions)
Pharm lyse, by BD (1 mentions)
Streptavidin hrp, by BioLegend (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Stain free imager, by Bio-Rad (1 mentions)
A0024, by Agilent Technologies (1 mentions)
Anti sod1 antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Odyssey machine, by LI COR (1 mentions)
8 protocols using anykd gradient gel
1
Analysis of CD11b and STAT5 Phosphorylation
2
Protein Extraction and Western Blotting
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Raji cells were plated at 105 cells ml−1 in 10 cm diameter dishes and dosed with the indicated treatment. Cells were washed with cold PBS and lysed for 10 min in lysis buffer (50 mM Tris–HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100) containing protease inhibitors (Complete, Roche), 1 mM PMSF and phosphatase inhibitors. Samples were clarified by centrifugation at 14 000 × g for 10 min, quantified with Bradford reagent (Bio-rad), denatured by boiling in Laemmli buffer (LI-COR) for 5 min and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using AnyKD gradient gels (Biorad). Protein was transferred to a PVDF membrane (Millipore) and blocked with Odyssey blocking buffer (LI-COR). Both primary antibodies and appropriate IR-dye conjugated secondary antibodies (LI-COR) were incubated in blocking buffer with 0.2% Tween. Anti-actin was used to control for equal loading and experiments were done in at least two independent biological replicates. Bands were visualized on a LI-COR Odyssey infrared imager.
3
Western Blot Analysis of CD200R1L in Neutrophils
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For western blot analysis of CD200R1L, neutrophils (both isolated by Ficoll and FACS sorted) were lysed in boiling modified 2× Laemmli buffer (125 mM Tris‐HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2‐mercaptoethanol, 0.004% bromophenol blue) for 5 min. Samples were loaded under denaturing conditions on AnyKD gradient gels (BioRad). Protein was transferred to PVDF membranes (Immobilon‐P PVDF 45um, Merck Chemicals BV). Membranes were blocked in 5% fat‐free milk (Elk, Campina, The Netherlands) in TBS 0.05% Tween‐20 (TTBS), and incubated overnight with CD200R1L‐biotin antibody (Sino Biological), anti‐MYC‐biotin antibody (Cell Signaling) or CD200R‐biotin (Serotec) in 1% Elk TTBS. Blots were washed and HRP labelled secondary antibody or streptavidin‐HRP (bioLegend) was added for 1 h at 4 °C. The blots were imaged using SuperSignal West Femto (Thermo Fisher Scientific) on a Bio‐rad Chemidoc MP.
4
Protein Extraction and Western Blotting
5
SOD1 Variant Electrophoresis Protocol
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Superoxide dismutase-1 (SOD1) variants at a concentration of 30 μM (10 μg total protein per well) were loaded into stain-free any kD gradient gels (Biorad, USA) in a tris-glycine buffer. Gels were electrophoresed at 100 V for 3 h at 4°C with stirring. Following electrophoresis, gels were imaged on a stain-free imager (Biorad, USA).
6
Tau and HspB8 Protein Analysis
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Triton-soluble and -insoluble fractions were diluted into 1X SDS sample buffer. Triton-soluble samples were incubated at 99 °C for 3 min and all samples were processed by SDS-PAGE in precast Any Kd gradient gels (BioRad; Hercules, CA, USA) and transferred to PVDF membranes. PVDF membranes were reversibly stained with Ponceau S to confirm proper transfer and equivalent loading of total protein in each lane. Rabbit anti-tau polyclonal antibody (Dako/Agilent, A0024; Santa Clara, CA, USA) was used to detect total tau protein. Goat anti-HspB8 (Hsp22) antibody (ThermoFisher, PA5-18103, directed to the CTD; Waltham, MA, USA) was used to detect Hsp22 protein.
7
Western Blot Analysis of SOD1
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Protein concentrations were determined using the 660 nm Protein Assay Reagent (Pierce). Protein samples in 1 × Laemmli buffer with 2-mercaptoethanol at 2.5% were boiled for 5 min. Equal amounts of protein for each condition were subjected to SDS-PAGE on an AnykD gradient gel (Bio-Rad) followed by immunoblotting with anti-SOD1 antibody (Cell Signaling) after wet transfer for 1 h to PVDF membrane (Bio-Rad) and blocking with non-fat dry milk.
8
Protein Extraction and Western Blot Analysis
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Protein was extracted by adding 10 mg of spinal cord tissue to 300 μL of RIPA buffer (Santa Cruz) and sonicating at 10 amps for 10 seconds followed by 1 h incubation on ice vortexing every 10 minutes. Samples were centrifuged for 30 minutes at 4°C and supernatants were collected. Samples of 25 μg of protein in Laemmli loading buffer were analyzed under reducing conditions (BioRad Any kD gradient gel, mini-Protein precast gel), and then transferred to nitrocellulose membranes. Membranes were stained for GAPDH (Cell Signaling, Rabbit) and CXCL10 (R&D, Goat) followed by staining with fluorescently tagged secondary antibodies and imaging using a LI-COR Odyssey machine.
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