Protein was collected from EVs, cells or cell lysates through lysis with radioimmunoprecipitation assay (RIPA) buffer supplemented with 1:100 Protease Inhibitor Cocktail Set III and Phosphatase Inhibitor Cocktail 1 & 2 (Sigma-Aldrich). Protein was quantified using a Pierce BCA Protein Assay Kit (ThermoFisher). Ten μg of protein was separated using NuPAGE 4%–12% Bis-Tris Mini Protein Gels (ThermoFisher) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked in 5% BSA, 1X TBS, and 0.1% Tween-20 at RT for 1 h. Membranes were then incubated overnight at 4°C with the appropriate primary antibody: 1:1000 diluted anti-Alix (sc-53540), anti-Hsp-70 (sc-24), anti-Flotillin-1 (sc-74566), anti-CD81 (sc-166029), anti-CD63 (sc-5275), and anti-GRP 94 (sc-393402) (all from Santa Cruz), and anti-TGFβ (#3711, Cell Signaling). After washing the membranes, they were subsequently incubated with peroxidase-conjugated 1:2000 Anti-Mouse (NXA931, GE Healthcare) or Anti-Rabbit (#7074, Cell Signaling). Detection was performed using Amersham ECL Western Blotting Detection Kit (GE Healthcare) and Chemidoc MP Imaging System (Bio-Rad).
Anti mouse nxa931
Manufactured by GE Healthcare
Sourced in United Kingdom
The Anti-mouse NXA931 is a laboratory reagent designed for use in various immunoassay and immunohistochemistry applications. It functions as a detection antibody that specifically binds to mouse immunoglobulins, allowing for the identification and visualization of target antigens in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail set 3, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 1 2, by Merck Group (1 mentions)
Nupage 4 12 bis tris mini protein gel, by Thermo Fisher Scientific (1 mentions)
Anti cd81 sc 166029, by Santa Cruz Biotechnology (1 mentions)
Anti cd63 sc 5275, by Santa Cruz Biotechnology (1 mentions)
Anti tgf β, by Cell Signaling Technology (1 mentions)
Amersham ecl western blotting detection kit, by GE Healthcare (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Actin a2066, by Merck Group (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
P erk thr202 tyr204, by Santa Cruz Biotechnology (1 mentions)
Complete protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Imagequant 4000 biomolecular imager, by GE Healthcare (1 mentions)
Hybond ecl nitrocellulose, by GE Healthcare (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Poly d lysine coated plates, by Merck Group (1 mentions)
4 protocols using anti mouse nxa931
1
Protein Isolation and Analysis from EVs, Cells, and Lysates
2
Western Blot Analysis of Protein Signaling
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Lysates from whole skin, epidermis, dermis or cultured cells were prepared as previously described7 (link) using buffers supplemented with Complete protease and phosphatase inhibitor cocktails (Merck). Protein concentrations were measured using Bradford reagent (BioRad, Hercules, California, USA) and 20–30 µg of protein/sample was boiled in Laemmli buffer, separated on SDS-PAGE, and transferred to Hybond ECL nitrocellulose (GE Healthcare, Illinois, USA). Nitrocellulose membranes were stained with Ponceau S (Merck) to verify equal protein loading and transfer prior to blocking and antibody incubations. Primary antibodies used were from Cell Signalling Technology (Danvers, Massachusetts, USA): p-GR Ser211, #4161S, 1/2000; p-ERK Thr202/Tyr204, #4376, 1/1000; p-p38 Thr180/Tyr182 #4631, 1/1000; p-JNK Thr183/Tyr185 #9251, 1/1000, and JNK #9252, 1/1000; Santa Cruz Biotechnology (Dallas, Texas, USA): GR, sc1004, 1/2000; Sigma: actin, A2066, 1/4000; and tubulin, T6199, 1/4000. Peroxidase-conjugated secondary antibodies were from GE Healthcare: anti-rabbit, NA934 and anti-mouse NXA931. Immunoreactive bands were detected using Pierce ECL Plus Western Blotting Substrate (ThermoFisher) and the ImageQuant 4000 Biomolecular Imager (GE Healthcare). Band intensities were quantitated using Image J software and were normalized to the loading controls, actin, or tubulin.
3
PTRN-1 Protein Detection and Co-Immunoprecipitation
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Worm protein samples were prepared by boiling 10 μl pellets of mixed-stage worms in SDS-mercaptoethanol solution. We detected PTRN-1 using antibody OD208A (Wang et al., 2015 (link)); we used the anti-actin antibody ACTN05 (Abcam, Cambridge, MA, C4) as a loading control. We used HRP-linked anti-rabbit IgG NA934V and anti-mouse NXA931 as secondary antibodies (GE Healthcare Lifesciences) at 1:1000 dilution in TBS, and added SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA). The blot result was replicated once.
To test co-immunoprecipitation, a 1:1 ratio of tagged DAPK-1 and PTRN-1 were co-transfected using Lipofectamine 2000 (Invitrogen) into HEK293 cells growing on poly-D-Lysine-coated plates (Sigma-Aldrich) in Opti-MEM (Thermo Fisher Scientific). After two days, cells were collected in cold PBS and lysed in lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP40, 0.5 mM EDTA, 3 mM MgCl2) for 30 min at 4°C. M2-FLAG conjugated magnetic beads (Sigma) were used for IP, and mouse anti FLAG (F1807, Sigma, 1:1000) and anti HA (HA-7, Sigma, 1:5000) antibodies were used for western blotting. The co-IP was repeated twice.
To test co-immunoprecipitation, a 1:1 ratio of tagged DAPK-1 and PTRN-1 were co-transfected using Lipofectamine 2000 (Invitrogen) into HEK293 cells growing on poly-D-Lysine-coated plates (Sigma-Aldrich) in Opti-MEM (Thermo Fisher Scientific). After two days, cells were collected in cold PBS and lysed in lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP40, 0.5 mM EDTA, 3 mM MgCl2) for 30 min at 4°C. M2-FLAG conjugated magnetic beads (Sigma) were used for IP, and mouse anti FLAG (F1807, Sigma, 1:1000) and anti HA (HA-7, Sigma, 1:5000) antibodies were used for western blotting. The co-IP was repeated twice.
4
Immunoblotting Analysis of K562 Cells
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