To quantify the expression levels of MEX-5 and MEX-5(ZFmut), 50 young adults were collected for each strain in 50 μl M9 buffer. 4× Laemmli sample buffer (Bio-Rad) was added to 1×, and samples were lysed by three freeze/thaw cycles on dry ice and in a 42° C water bath. DTT was then added to a final concentration of 200 mM before boiling at 98° C for 5 min. Equal volumes of each sample were loaded onto Any kD mini-PROTEAN TGX Gels (Bio-Rad). Samples were transferred to Immobilon-P transfer membrane (EMD Millipore) and probed with 1:500 guinea pig anti-MEX-5 primary antibody [13 (link)] and 1:10,000 peroxidase-conjugated AffiniPure Goat anti-guinea pig IgG secondary antibody (Jackson ImmunoResearch). The blot was then stripped with Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) and probed with 1:600 mouse monoclonal anti-α-tubulin primary antibody DM1A (Sigma-Aldrich) and 1:5000 peroxidase-conjugated AffiniPure Goat anti-mouse IgG secondary antibody (Jackson Immuno Research). All antibodies were dissolved in TBST containing 5% milk. Blots were developed with the Clarity Western ECL Substrate (Bio-Rad) and imaged with the ChemiDoc XRS system (Bio-Rad) with an exposure time of 20 seconds.
Affinipure goat anti mouse igg secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
AffiniPure goat anti-mouse IgG secondary antibody is a laboratory reagent used to detect and quantify mouse immunoglobulin G (IgG) in various immunological assays. It binds specifically to the Fc region of mouse IgG, allowing for the identification and measurement of mouse IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p transfer membrane, by Merck Group (1 mentions)
Restore plus western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Anykd mini protean tgx gels, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Glutaraldehyde, by Science Services (1 mentions)
Formaldehyde, by Science Services (1 mentions)
Anti cd63 ab59479, by Abcam (1 mentions)
Sis veleta ccd camera, by Olympus (1 mentions)
Tecnai g2 spirit biotwin, by Olympus (1 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Sc 32233, by Santa Cruz Biotechnology (1 mentions)
4 protocols using affinipure goat anti mouse igg secondary antibody
1
Quantifying MEX-5 Protein Expression Levels
2
Transmission Electron Microscopy of Extracellular Vesicles
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EV‐rich fractions (8–9) were subjected to transmission electron microscopy to assess the presence and morphology of EV. A drop (3 µL) of sample was adhered to pre‐coated formvar/carbon/glow discharged 100 mesh copper‐palladium grids for 20 min. Grids were fixed with 2% formaldehyde (Science Services) in 0.1 m phosphate buffer for 20 min, washed with sterile water and stained with 2% uranyl acetate for 5 min. For immunogold labeling, after fixation, the grids were immersed in 50 × 10−3 m glycine in PBS for 15 min and blocked for 10 min with 10% fetal calf serum (FCS) in PBS. Without rinsing, the grids were immediately placed into the primary antibody (1:200 diluted in 5% FCS in PBS, anti‐CD63 ab59479, Abcam) for 30 min at RT. As control, some of the grids were not exposed to the primary antibody. Grids were rinsed with 0.5% FCS in PBS and incubated with 12 nm colloidal gold‐AffiniPure goat‐anti‐mouse IgG secondary antibody (1:20 diluted in 5% FCS in PBS, 115‐205‐146, Jackson Immunoresearch) for 30 min at RT. The grids were rinsed with PBS and post‐fixed in 1% glutaraldehyde (Science Services) for 5 min. After rinsing in distilled water, the grids were stained using 2% uranyl acetate for 5 min. Grids were examined using electron microscopy (FEI Tecnai G2 Spirit BioTWIN operating at 120 keV equipped with an Olympus‐SIS Veleta CCD Camera).
3
Western Blot Analysis of MLSN Protein
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Proteins from cell lysates were separated by 8% sodium dodecyl sulfate gel electrophoresis, transferred onto nitrocellulose membranes, and then probed with the MLSN mAb primary antibody (1:500; ab133489; Abcam, USA) for 24 h at 4°C. This procedure was followed by incubation with horseradish peroxidase-conjugated AffiniPure goat anti-mouse IgG secondary antibody (115-035-008; Jackson ImmunoResearch) for 1 h at room temperature.
4
Western Blot Analysis of GALNT14
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Western blotting was performed using 50 µg of total protein from culture cells as described previously [27 (link)]. Depending on size, proteins were resolved on 7.5–12.5% polyacrylamide gel. The resolved proteins were transferred to 0.22 µm poly (vinylidene fluoride) (PVDF) membranes and blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature (RT). The membranes were incubated with primary antibodies against GALNT14 (1:1000, sc-393051, SANTA CRUZ, Dallas, Texas, USA) and GAPDH (1:10,000, sc-32233, SANTA CRUZ) overnight at 4 °C. The membranes were then incubated with a peroxidase AffiniPure goat anti-mouse IgG secondary antibody (1:2000, 115-035-003, Jackson ImmunoResearch, Bar Harbor, ME, USA) for 2 h at RT, and visualized using the SuperSignal West Femto chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA). GAPDH was used as an internal control. The results were quantitated using ImageJ software.
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