In Vitro Secretion of Xanthomonas Effectors

For in vitro secretion, ORFs of XC2081 (avrBs1), XC3176, and XC3002 (hpa1) were cloned into a host-broad plasmid pJXG containing a 3×FLAG-encoding sequence [18 (link)], respectively. Bacteria of Xcc were cultured in rich NYG medium overnight and were transferred into hrp-inducing medium MMX at same cell density for 8 h. The total cell extracts and cultural supernatants were isolated as described previously [31 (link)]. Equal amounts of total cell extract and cultural supernatants were separated by SDS-PAGE and analyzed by immunoblotting, using anti-Flag-tag rabbit monoclonal antibody (1/1000, Sigma). Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (1/1000, Pierce) was used as secondary antibody. To ensure that no bacterial lysis had occurred, the protein blots were routinely probed with a rabbit monoclonal antibody specific for β subunit of E. coli RNA polymerase (RNPβ, 1/1000, Abcam, ab12087). Reactions were visualized by enhanced chemiluminescence. All experiments were repeated at least twice.

Free full text: Click here

Gan Y.L., Yang L.Y., Yang L.C., Li W.L., Liang X.L., Jiang W., Jiang G.F., Hang X.H., Yang M., Tang J.L, & Jiang B.L. (2021). The C-terminal domain of the type III secretion chaperone HpaB contributes to dissociation of chaperone-effector complex in Xanthomonas campestris pv. campestris. PLoS ONE, 16(1), e0246033.

Publication 2021

anti-Flag-tag rabbit monoclonal antibody ab12087

Anti antibody Antibody Bacterial Cell extract Chemiluminescence E coli Goat Horseradish peroxidase Hpa1 Immunoglobulin g Monoclonal antibody Orfs Plasmid Protein Rabbit Sds page Secretion β subunit of rna polymerase

Corresponding Organization : Guangxi University

Top 5 similar protocols

Variable analysis

independent variables

ORFs of XC2081 (avrBs1), XC3176, and XC3002 (hpa1) cloned into a host-broad plasmid pJXG containing a 3×FLAG-encoding sequence

dependent variables

Secretion of proteins XC2081 (avrBs1), XC3176, and XC3002 (hpa1) into the cultural supernatants

control variables

Bacterial culture conditions (NYG medium overnight, then transferred to hrp-inducing medium MMX at same cell density for 8 h)
Protein detection method (SDS-PAGE, immunoblotting using anti-Flag-tag antibody and secondary antibody)
Checking for bacterial lysis (probing with antibody specific for E. coli RNA polymerase β subunit)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

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?

Revolutionizing how scientists
search and build protocols!