Cell extracts were prepared in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 150 mN NaCl, 0.1% TritonX-100 and protease and phosphatase inhibitors (Boehringer Mannheim) and protein content was assayed by the Bradford method (BioRad) using a bovine serum albumin standard curve as described earlier [21 (link), 22 (link)]. Lysates containing 10–20μg total protein per lane were resolved by SDS-PAGE, electrostatically transferred to polyvinylidene difluoride membrane and blocked with Odyssey Blocking Buffer (Li-COR Biosciences, Nebraska USA). The blots were probed with the indicated primary antibodies in TTBS (50 mM Tris, 150 mM NaCl, 0.05% Tween 20) followed by incubation with the secondary antibody, IRDye 800 goat anti-mouse or IRDye 680 Goat anti-Rabbit IgG (1:15000, Li-COR Biosciences) as applicable and resultant bands was visualized using the Odyssey Infrared Imaging System, v3.0 (Li-COR Biosciences, Nebraska USA). Primary antibodies used were as follows: inducible HSP70 (HSPA1A) (1:10000; SPA-812, Enzo), total HSP70 (1:5000; H5147, Sigma), CD63 (1:1000; SantaCruz), β-tubulin (1:10000; Chemicon), and total and phospho-p38 (both 1:1000; Cell Signaling).
Spa 812
Manufactured by Enzo Life Sciences
Sourced in United States
The SPA-812 is a highly sensitive and versatile luminescence detection system designed for a wide range of life science applications. It features a compact and ergonomic design, providing researchers with a reliable and efficient tool for their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Spa 810, by Enzo Life Sciences (2 mentions)
Goat anti mouse irdye 800, by LI COR (1 mentions)
Irdye 680 goat anti rabbit igg, by LI COR (1 mentions)
Total and phospho p38, by Cell Signaling Technology (1 mentions)
β tubulin, by Merck Group (1 mentions)
H5147, by Merck Group (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Adi spp 715, by Enzo Life Sciences (1 mentions)
Adi spa 800, by Enzo Life Sciences (1 mentions)
Adi spa 803, by Enzo Life Sciences (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Bovine serum albumin (bsa), by Roche (1 mentions)
Superblock solution, by ScyTek Laboratories (1 mentions)
Rabbit normal blocking serum, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg fitc, by Abcam (1 mentions)
D eclipse c1, by Nikon (1 mentions)
Triton x 100, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
5 protocols using spa 812
1
Western Blot Analysis of Protein Expression
2
Antibody and protein detection protocol
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HSP70 monoclonal antibody (SPA-810), HSP27 monoclonal antibody (ADI SPA-800), human recombinant HSP70 protein (SPP-755), polyclonal HSP70 antibody (SPA-812), recombinant human HSP27 (ADI-SPP-715), and polyclonal HSP27 antibody (ADI- SPA-803), were from Enzo Lifesciences (Antwerp, Belgium). Ficoll was from Nycomed (Oslo, Norway), Bovine serum albumin (BSA) was from Roche (Boehringer Mannheim, Germany), Fetal calf serum was from Biochrom (International Medical, Wavre, Belgium), RIPA buffer was made up of 1% nonidet P40, 1% deoxycholic acid, NaCl 150 mM, 0.1% SDS, 1% Triton X-100, phosphate inhibitors (50 mM NaF, 10 mM h-glycerophosphate, 1 mM Na2P2O7 10H2O, 10 mM Na3VO4, 10 mM p-nitrophenylphosphate) and a cocktail of protease inhibitors (10:1000). Fix and Perm cell permeabilization kit was from IMTEC (Antwerp, Belgium). Phosphate-buffered saline (PBS) consisted of 40 mM Na 2HPO4 2H2O, 135 mM NaCl, 3 mM KCl, and 1 mM KH2PO4.
3
Immunohistochemical Analysis of Heat Shock Proteins
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SYF sections were boiled in sodium citrate buffer (0.05% Tween-20 in 0.01 M sodium citrate, pH 6.0) for 10 min to enhance antigen retrieval. Endogenous peroxidase activity was quenched with 3% H2O2 for 10 min and nonspecific binding was blocked with super block solution (ScyTek Laboratories, West Logan, UT, USA) for 20 min. Then, sections were incubated with 1:100 diluted anti-HSP70 rabbit polyclonal antibody (SPA-812; EnZo Life Science) or anti-HSP25 mouse polyclonal antibody (IAP-28; Abcam) at room temperature for 1 h. After washing three times in PBST (0.05% Tween-20 in PBS) for 5 min, sections were incubated with goat anti-rabbit IgG (H + L) secondary anti-body–Alexa Fluor 488 (A-11008; Thermo Fisher Scientific, Waltham, MA, USA), or goat anti-mouse IgG (H + L) secondary anti-body–Alexa Fluor 546 (A-11003; Thermo Fisher Scientific) for 30 min. After washing with PBST three times, the cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI). To confirm the specificity of the antibodies, negative controls were subjected to the same procedure, except that the primary antibody was replaced by mouse, rat, or rabbit IgG (Vector Laboratories, Burlingame, CA, USA).
4
Antibody Characterization for PRRSV
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The mouse anti-HSP70 MAb (SPA-810) and rabbit polyclonal anti-HSP70 antibody (SPA-812) were obtained from Enzo Life Sciences (Farmingdale, NY, USA), and the rabbit anti-β-actin MAb (13E5) was obtained from Cell Signaling Technology (Beverly, MA, USA). The anti-PRRSV N protein MAb was obtained from Jeno Biotech Inc (Chuncheon, South Korea). The mouse monoclonal antibody (J2) specific for dsRNA was purchased from Scicons (Hungary).
5
Intracellular Colocalization of AGO2 and HSP70
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For immunofluorescence analysis, cells were transferred from U-bottom plates to gelatin-coated microscope slides by cytospine (300xg, 10 min) and perfused by PBS containing 4% (w/v) paraformaldehyde for 20 min at room temperature. Fixed cells were washed with PBS and blocked with 10% rabbit normal blocking serum (Santa Cruz Biotechnology, Dallas, TX, USA), supplemented with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 45 minutes at RT. Next, cells were washed and double-stained for HSP70 (rabbit polyclonal antibody, 1:500, SPA-812, Enzo Life Sciences, Inc., Farmingdale, NY, USA) and AGO2 (mouse monoclonal antibody, 1:500, B-3, Santa Cruz Biotechnology, Dallas, TX, USA) with PBS supplemented with 1.5% of normal blocking rabbit serum and 0.3% Triton X-100, overnight at 4 °C in wet chamber. Subsequently, cells were washed and secondary fluorescent antibody (goat anti-rabbit IgG-TR and goat anti-mouse IgG-FITC; 1:100 both Abcam, Cambridge, UK), supplemented with 1.5% of normal blocking rabbit serum and 0.3% Triton X-100 was added for 1 h at RT. For fluorescent DNA nuclei staining, DAPI was used (1.5 mg/mL UltraCruz Mounteining Medium, Santa Cruz Biotechnology, Dallas, TX, USA). The intracellular colocalization of AGO2 and HSP70 was analyzed by confocal microscopy (Nikon D-Eclipse C1) using EZ-C1 software.
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