Whole-cell lysates were prepared from age-matched healthy controls and patient-derived T cell lymphoblasts. Cell lysates from one million cells per lane or 80 µg of protein were loaded on 10% polyacrylamide gel and separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane using the Trans-Blot Turbo System (Bio-Rad). Blots were probed overnight with specific primary antibodies—for instance, polyclonal anti-human SLP-76 previously described (Gonen et al., 2005 (link)), anti-human SLP76 (ab17029; Abcam), anti–PLC-γ1 (sc-81; Santa Cruz), anti–phospho-PLC-γ Tyr783 (AT-7142; MBL), anti-human phosphor-MAPK (T202\Y204-Erk1/2; #9101; Cell Signaling Technology), and anti-human p44/42 MAPK-Erk1/2 (#9102; Cell Signaling Technology). Stimulation was induced with anti-CD3 (10 µg/ml OKT; Invitrogen). Anti-HSC70 (sc-7298; Santa Cruz Biotechnology) was used as a loading control. For imaging, appropriate HRP-labeled secondary antibodies (1:10,000) were used. Bands were revealed using enhanced chemiluminescence (Biological industries). Densitometry was performed on scanned immunoblot images using ImageJ software.
Anti hsc70 sc 7298
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-HSC70 (sc-7298) is a primary antibody product offered by Santa Cruz Biotechnology. It is designed to detect the Heat Shock Cognate 70 kDa Protein (HSC70), a constitutively expressed member of the heat shock protein 70 family.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence, by Sartorius (1 mentions)
Anti plcγ1 sc 81, by Santa Cruz Biotechnology (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Anti actin a5441, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Anti rabbit 32460, by Thermo Fisher Scientific (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti aqp4 ab3594, by Merck Group (1 mentions)
4 protocols using anti hsc70 sc 7298
1
Immunoblot Analysis of T cell Signaling
2
Protein Stability Assay in HPV-Transduced Cells
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HaCaT cells transduced with HPV16 E6, E7 or firefly luciferase (negative control) were treated with 100 µM Cycloheximide (Sigma Aldrich). The proteins were isolated in SDS buffer (SDS 1%, Tris-HCl pH 7.5 40 mM, EDTA 1 mM, protease inhibitors) at different time points (up to 24 h) and then quantified (BCA protein assay; Pierce). The level of given proteins was finally determined by western blot (see Table S3 for primary antibody specificities). Anti-actin (A5441; Sigma-Aldrich) and anti-HSC-70 (sc-7298; Santa Cruz Biotechnology) antibodies were used for normalization.
3
Regulation of mTOR and HIF-1α Signaling
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Rapamycin was dissolved in DMSO. The final concentration of DMSO in the culture medium did not exceed 0.2%. Anti-phospho-mTOR (ab109268, diluted 1:2,000) and anti-HIF-1α (ab51608, diluted 1:2,000) were from Abcam (Cambridge, UK). Anti-phospho-Akt (#4058, diluted 1:1,000) was from Cell Signaling Technology (Beverly, MA, USA) and anti-HSC70 (sc7298, diluted 1:10,000) was from Santa Cruz biotechnology (Dallas, TX, USA). Peroxidase-conjugated anti-mouse (#32430, diluted 1:2,000) and anti-rabbit (#32460, diluted 1:2,000) secondary antibody were from ThermoScientific (Waltham, MA, USA). Lysis buffer: [50 mM Hepes (pH 7.5), 150 mM sodium chloride, 1 mM EDTA (pH 8), 2.5 mM EGTA (pH 7.4), 0.1% Tween 20, 10% glycerol, 0.1 mM sodium orthovanadate, 1 mM sodium fluoride and 10 mM β-glycerophosphate] plus Protease inhibitor cocktail (#539134 Calbiochem, Darmstadt, Germany), PMSF and Phosphatase inhibitor Cocktail Set II (#524636 Calbiochem).
4
Quantifying Aqp4 Protein in Rat Stroke
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For quantification of Aqp4 protein abundance in rat tissues, brains from rats submitted to 120 minutes MCAO were harvested after 48 hours reperfusion. Brains were isolated and 3 mm coronal slabs were cut; a slab that bridged from approximately 1 mm anterior to bregma to −2 mm posterior to bregma was isolated. Coronal slabs from control and from rats submitted to MCAO were stained with 2 % triphenyl tetrazolium chloride (TTC) dissolved in 0.9 % saline for 20 minutes at room temperature to determine the location of the ischemic core and penumbra; this technique does not affect the quality of isolated RNA or protein [53 (link)]. The necrotic tissue (white) was removed and discarded (the core was not analyzed due to protein degradation) whereas the surrounding cortical penumbra (red) and the ipsilateral subcortical white matter (lateral corpus callosum and external capsule) were dissected, homogenized in lysis buffer (1 % Triton X-100 in 1 x dPBS), and analyzed by immunoblot. Proteins were detected using anti-Aqp4 (AB3594; Millipore) and anti-Hsc-70 (sc-7298; Santa Cruz Biotechnology, Inc., Dallas, TX).
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