Lysates from cells and tissues were subjected to 10 % SDS-PAGE (Bio-Rad, Pre-gel, MA); separated proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Seoul, Korea). Membranes were blocked with TBS-based blocking buffer (Li-COR Biosciences, Lincoln, NE) for 1h at room temperature, and incubated overnight at 4 °C with primary antibodies p16, SOD1,SOD2, ERK, p-ERK, CREB, p-CREB, p-elF2α, Beclin1, Bip, ATG12, LC3B, p62, BNIP3, PINK1, Parkin, PPARα, FATP4, Tau, P-tau, Beta-amyloid, GFAP, iba-1, beta-actin (1:1000, Cell Signaling Technology, Danvers, MA). Membranes were then washed with TBST three times for 10 min each and incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000, Cell Signaling Technology, Danvers, MA) for 1 h at room temperature and washed again. Signals were detected by Odyssey-LC chemiluminsescent imaging system (LI-COR, Lincoln, NE). Signals were averaged and expressed as described in figure legend.
Tbs based blocking buffer
Manufactured by LI COR
Sourced in United States
TBS-based blocking buffer is a reagent used in various immunoassay techniques to reduce non-specific binding and improve the signal-to-noise ratio. It is a commonly used solution for blocking unoccupied binding sites on solid supports or membranes, thereby minimizing background signals during the detection step of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Bnip3, by Cell Signaling Technology (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Iba 1, by Cell Signaling Technology (1 mentions)
P elf2α, by Cell Signaling Technology (1 mentions)
Beta amyloid, by Cell Signaling Technology (1 mentions)
Odyssey lc chemiluminsescent imaging system, by LI COR (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions)
Pparα, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
P tau, by Cell Signaling Technology (1 mentions)
Parkin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab109390, by Abcam (1 mentions)
Mab361, by Merck Group (1 mentions)
Image lab software version 6, by Bio-Rad (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
3 protocols using tbs based blocking buffer
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Protein Samples
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Cell lysates containing 1× SDS buffer were homogenized with a 27-G syringe and whole lysates were run on 4% to 20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, 4568096) or 6/10/12% home-made polyacrylamide gels before being transferred to PVDF membranes (Bio-Rad, 1620177). Membranes were then washed in TBS-T before being blocked with TBS-based blocking buffer (LI-COR). Membranes were incubated with primary antibodies and then washed again before being incubated with species-specific secondary antibodies and imaged using LI-COR imaging.
3
Western Blot Analysis of Phospho-Tau
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Proteins were loaded onto a 8% SDS-Page gel at a concentration of 10 μg and were separated by gel electrophoresis (Laemmli, 1970). Afterwards, the proteins were transferred onto a polyvinylidene difluoride transfer membrane (Cat. No. IPVH00010, Millipore-Sigma, MO, USA) followed by TBS-based blocking buffer (LI-COR, NE, USA) gently shaking overnight at 4 °C. On the following day, the membranes were incubated with primary antibody (rabbit anti-phospho-tau (pTau 396), 1:5000, Abcam ab109390; mouse anti-tau-5 (total tau), 1:5000, Millipore MAB361) gently shaking overnight in 4 °C. On the next day, the membranes were washed in 1×TBS + 0.1% Tween-20, followed by 1×TBS washes, and then incubated in IRDye near-infrared secondary antibodies (1:5000) for 1 h at room temperature. Lastly, the membranes were washed in 1×TBS + 0.1% Tween-20, followed by 1×TBS washes, and then imaged on ChemiDoc MP Imaging System (BIO-RAD, CA, USA). Target band intensity signals were quantified utilizing the Image Lab Software version 6.0 (BIO-RAD, CA, USA). Normalization of the target bands was completed using anti-total tau signals.
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