Equal amounts of lysates were loaded onto Mini-Protean TGX (4-20%) SDS-PAGE gels (BioRad). All blots except phospho-MTFR1L were transferred to nitroceullose membranes (BioRad) with a Trans-Blot Turbo and blocked for one hour in Intercept Buffer (Li-COR). Membranes were then incubated in their respective primary antibodies in blocking buffer at 4°C overnight. Blots probing for phospho-MTFR1L we transferred to polyvinylidene difluoride membranes (PVDF, Immobilion) and blocked in 5% fat-free dry milk in TBS-T for 1 hour before primary antibody incubation for four days at 4°C. Membranes were incubated in Li-Cor fluorescence-coupled secondary antibodies for 1 hour at room temperature prior to visualization with a Li-Cor Odyssey Blot Imager.
Trans blot turbo
Manufactured by LI COR
The Trans-Blot Turbo is a semi-dry electrophoretic transfer system designed for the rapid and efficient transfer of proteins from polyacrylamide gels to membranes. It is capable of performing high-quality protein transfers in a matter of minutes.
Automatically generated - may contain errors
Lab products found in correlation
Nitroceullose membranes, by Bio-Rad (2 mentions)
Mini protean tgx 4 20 sds page gel, by Bio-Rad (2 mentions)
Odyssey blot imager, by LI COR (2 mentions)
Intercept buffer, by LI COR (2 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Merck Group (1 mentions)
P creb, by Cell Signaling Technology (1 mentions)
Anti taz, by BD (1 mentions)
Intercept pbs blocking buffer, by LI COR (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Mouse anti prx2, by Abcam (1 mentions)
Rabbit anti prx3, by Abcam (1 mentions)
Tris glycine sds page gel, by Bio-Rad (1 mentions)
Odyssey clx system, by LI COR (1 mentions)
Goat anti mouse irdye 680, by LI COR (1 mentions)
Goat anti rabbit irdye 800, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
5 protocols using trans blot turbo
1
Western Blotting Protocol for Protein Analysis
2
Quantitative Western Blotting Protocol
3
Western Blot Analysis of Protein Samples
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Cell pellets were resuspended in protein sample buffer (125 mM Tris–HCl pH 6.8, 10% Glycerol, 1% SDS, 0.2% (w/v) Orange G, 5% β‐mercaptoethanol) and incubated in 98°C for 5 min. Benzonase (50 U) was added to the lysates and incubated at room temperature for 30 min, followed by incubation at 98°C for 5‐ and 10‐min centrifugation. Soluble extracts were separated on a 10% or 12% SDS–PAGE gel (BioRad), then transferred to nitrocellulose membranes (BioRad Transblot Turbo), blocked with Intercept (PBS) Blocking Buffers (LI‐COR) for 1 h and incubated overnight with primary antibodies at 4°C. The following day, blots were washed three times with TBST (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Tween‐20) and incubated with secondary antibodies for 1 h, with subsequent three times TBST washing. The blots were analyzed by Odyssey Imaging System (LI‐COR) and quantified using ImageStudio.
4
Western Blot Analysis of Antioxidant Proteins
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Samples were resuspended in 4X loading buffer (200 mM Tris-Cl, pH (6.8), 8 % (w/v) SDS, 0.4 % (w/v) bromophenol blue, 40 % (v/v) glycerol), 400 mM DTT as a reductant. Typically 10-25 μg protein was loaded onto 12 % or 4 – 20% Tris-Glycine SDS-PAGE gels (BioRad) and run at 120 V for 45 min. Protein was transferred to PVDF membranes (Immobilon®-FL or BioRad Trans-Blot® Turbo™Mini) by wet transfer (in the presence of 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH (8.4)) or semi-dry transfer (BioRad Trans-Blot® Turbo™) before blocking for 1 h at RT with Odyssey blocking buffer (LICOR). Typically, primary antibody incubation was completed in 4 % (v/v) Odyssey buffer in PBS + 0.1 % (v/v) Tween-20 (PBST) for 1 h at RT. Membranes were then washed 3 x 15 min in PBST, followed by secondary incubation in 4 % Odyssey buffer in PBS for 1 h at RT, washed 2 x 15 min in PBST and 1 x 15 min in PBS. Membranes were then visualised using a LICOR Odyssey CLx system and analysed using ImageStudio Lite. Primary antibodies: rabbit anti-TPP (1:1000) (Lin et al., 2002 (link)), rabbit anti-TrxR (1:1000, Abcam), rabbit anti-Prx3 (1:500, Abcam), mouse anti-Trx (1:1000, Abcam) mouse anti-Prx2 (1:4000 Abcam). Secondary antibodies were goat anti-rabbit IRDye800 (1:25000, LICOR) and goat anti-mouse IRDye680 (1:25000, LICOR).
5
Western Blot Protocol
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Equal amounts of lysates were loaded onto Mini-Protean TGX (4-20%) SDS-PAGE gels (BioRad). All blots except phospho-MTFR1L were transferred to nitroceullose membranes (BioRad) with a Trans-Blot Turbo and blocked for one hour in Intercept Buffer (Li-COR). Membranes were then incubated in their respective primary antibodies in blocking buffer at 4 °C overnight. Blots probing for phospho-MTFR1L we transferred to polyvinylidene difluoride membranes (PVDF, Immobilion) and blocked in 5% fat-free dry milk in TBS-T for 1 hour before primary antibody incubation for four days at 4 °C. Membranes were incubated in Li-Cor fluorescence-coupled secondary antibodies for 1 hour at room temperature prior to visualization with a Li-Cor Odyssey Blot Imager.
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