Cells were grown at 30 °C in M9-based medium with 20 μg/ml each of the 20 amino acids, 2 μg/ml thiamine, and 0.4% glucose until mid-log phase. Total cellular proteins were precipitated with 5% trichloroacetic acid, washed with acetone, and dissolved in SDS sample buffer. Immunoblotting was carried out essentially as described previously (90 (link), 91 (link)). Proteins were separated by SDS-PAGE and electroblotted onto an Immobilon-P membrane filter (MilliporeSigma). Only when 15% bis-Tris gel was used for SDS-PAGE, a transferred membrane filter was dried at 37 °C for 30 min and then hydrophilized with methanol. After blocking with BLOTTO (90 (link)), the filter was incubated with an appropriate antibody. For anti-RseP immunoblotting, anti-RseP antibodies were preincubated with whole-cell lysates of AD1840 (the ΔrseA ΔrseP ΔdegS strain) at 4 °C for 1 h to reduce a background as described previously (82 (link)). The filter was then washed and incubated with goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxide (Bio-Rad). After washing of the filter, proteins that reacted with secondary antibodies were visualized using ECL or ECL Prime Western Blotting Detection Reagents (Cytiva) and Bio image analyzer LAS4000mini (Cytiva).
Goat anti mouse or anti rabbit igg conjugated with horseradish peroxide
Manufactured by Bio-Rad
Goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase is a secondary antibody used in immunoassays and immunoblotting techniques. It binds to the primary antibody and the attached horseradish peroxidase enzyme can be used to detect and quantify the target antigen.
Automatically generated - may contain errors
3 protocols using goat anti mouse or anti rabbit igg conjugated with horseradish peroxide
1
Immunoblot Analysis of RseP Protein
2
Immunoblotting for Myc, FtsH, and HflKC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown, and the total cellular proteins were precipitated with 5% trichloroacetic acid, washed with acetone, and dissolved in SDS sample buffer. Immunoblotting was carried out essentially as described previously (Yokoyama et al., 2021) (link). Proteins were separated by SDS-PAGE and electroblotted onto an Immobilon-P membrane filter (Millipore Sigma). After blocking, the filter was incubated with an appropriate antibody. Monoclonal anti-Myc antibody (c-Myc (9E10), Santa Cruz Biotechnology), rabbit polyclonal anti-FtsH antibody (Kihara et al., 1996) (link), and anti-HflKC antibody (Kihara et al., 1998) (link) were used for immunoblotting. For anti-HflKC immunoblotting, anti-HflKC antibodies were preincubated with whole-cell lysates of AK1136 (the DhflKC strain) at 4 C for 1 h to reduce the background. The filter was then washed and incubated with goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxide (Bio-Rad). After washing the filter, proteins that reacted with secondary antibodies were visualized using ECL or ECL Prime Western blotting Detection Reagents (Cytiva) and a mini LAS4000 Bio image analyzer (Cytiva) or an LAS3000 (Cytiva) instrument.
3
Immunoblotting Analysis of RseP Protease
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown at 30°C in M9-based medium with 20 μg/mL each of the 20 amino acids, 2 μg/mL thiamine and 0.4% glucose until mid-log phase. Total cellular proteins were precipitated with 5% trichloroacetic acid (TCA), washed with acetone and dissolved in SDS sample buffer. Immunoblotting was carried out essentially as described previously (90, 91) . Proteins were separated by SDS-PAGE and electroblotted onto an Immobilon-P membrane filter (MilliporeSigma). Only when 15% bis-Tris gel was used for SDS-PAGE, a transferred membrane filter was dried at 37°C for 30 min and then hydrophilized with methanol. After blocking with BLOTTO (90), the filter was incubated with an appropriate antibody. For anti-RseP immunoblotting, anti-RseP antibodies were pre-incubated with whole-cell lysates of AD1840 (the ΔrseA ΔrseP ΔdegS strain) at 4°C for 1 h to reduce a background as described previously (83) . The filter was then washed, and incubated with goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxide (Bio-Rad). After washing of the filter, proteins that reacted with secondary antibodies were visualized using ECL or ECL Prime Western Blotting Detection Reagents (Cytiva)
and Bio image analyzer LAS4000mini (Cytiva).
and Bio image analyzer LAS4000mini (Cytiva).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!