Proteins were isolated using NaOH/TCA and were resuspended in HU buffer (8 M Urea, 5% SDS, 200 mM Tris pH 6.8, 100 mM DTT). Proteins were resolved on 12% SDS-PAGE gels and transferred to PVDF membrane using a semi-dry transfer apparatus (BioRad). Membranes were blocked in 5% milk/PBST for ~30 minutes at room temperature followed by incubation with primary antibody overnight at 4°C. After washing with PBST, the membrane was incubated with the appropriate HRP-conjugated secondary antibody for ~1hr at room temperature before washing 3–4 × with PBST. Detection was carried out on a GE ImageQuant LAS 4000 using Pierce SuperSignal West Pico Chemiluminescent Substrate. The following antibodies were used: mouse anti-FLAG [M2] from Sigma-Aldrich; HA-probe antibody, Y-11 (Santa Cruz, sc-805), Anti-Renilla Luciferase Antibody, clone 1D5.2 (milipore, MAB4410), Anti-Firefly Luciferase antibody (Abcam, ab21176) rabbit anti-eRF1 was a gift from R. Green (Eyler et al., 2013 (link)); goat anti-mouse IgG HRP (31430) and goat anti-rabbit IgG HRP (31460) from Thermo Scientific.
Goat anti mouse igg hrp 31430
Manufactured by Thermo Fisher Scientific
Goat anti-mouse IgG HRP (31430) is a secondary antibody used to detect and quantify mouse immunoglobulin G (IgG) in various immunoassay applications. It is conjugated with horseradish peroxidase (HRP), which serves as a reporter enzyme for signal detection.
Automatically generated - may contain errors
3 protocols using goat anti mouse igg hrp 31430
1
Immunoblotting of Recombinant Proteins
2
Western Blot Protein Detection Protocol
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Proteins were resolved on 15% SDS PAGE gels and transferred to PVDF membranes using a semi-dry transfer apparatus (BioRad). The membranes were blocked with milk in PBST for ~ 30 minutes at room temperature followed by incubation with primary antibody overnight at 4°C. After washing with PBST, the membrane was incubated with the appropriate HRP-conjugated secondary antibody for ~ 1hr at room temperature before washing 3–4 × with PBST. Detection was carried out on a GE ImageQuant LAS 4000 using the Pierce SuperSignal West Pico Chemiluminescent Substrate. The following antibodies were used: mouse anti-PGK1[22C5D8] (ab113687) and rabbit anti-rpS9 (ab117861) from Abcam; rabbit anti-ASC1 was a gift from Wendy Gilbert (Yale University) [61 (link)]; mouse anti-rpL4 was a gift from Heather True (Washington University in St. Louis); goat anti-mouse IgG HRP (31430) and goat anti-rabbit IgG HRP (31460) from Thermo Scientific.
3
Immunoblotting of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were isolated using NaOH/TCA and were resuspended in HU buffer (8 M Urea, 5% SDS, 200 mM Tris pH 6.8, 100 mM DTT). Proteins were resolved on 12% SDS-PAGE gels and transferred to PVDF membrane using a semi-dry transfer apparatus (BioRad). Membranes were blocked in 5% milk/PBST for ~30 minutes at room temperature followed by incubation with primary antibody overnight at 4°C. After washing with PBST, the membrane was incubated with the appropriate HRP-conjugated secondary antibody for ~1hr at room temperature before washing 3–4 × with PBST. Detection was carried out on a GE ImageQuant LAS 4000 using Pierce SuperSignal West Pico Chemiluminescent Substrate. The following antibodies were used: mouse anti-FLAG [M2] from Sigma-Aldrich; HA-probe antibody, Y-11 (Santa Cruz, sc-805), Anti-Renilla Luciferase Antibody, clone 1D5.2 (milipore, MAB4410), Anti-Firefly Luciferase antibody (Abcam, ab21176) rabbit anti-eRF1 was a gift from R. Green (Eyler et al., 2013 (link)); goat anti-mouse IgG HRP (31430) and goat anti-rabbit IgG HRP (31460) from Thermo Scientific.
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