Western Blot Analysis of Toxoplasma Proteins

Tachyzoites were collected, filtered, counted and lysed by 6 cycles of rapid freezing/defreeze in hypotonic buffer, and boiled with LB for 5 minutes. 0.5 to 1×10⁷ parasites were loaded per well and resolved by 15% SDS-PAGE. Proteins were transferred to PVDF membrane for 1h at 100V. Western blot was then performed as described [90 (link)]. The primary antibodies: αrH2B.Z [22 (link)], αrH2A.Z [23 (link)], αrROP5 (rabbit) and αrROP18 (rabbit) were used at 1/5000, whereas α-c-Myc (rabbit, abCam ab9106) was used at 1/2000 and αrSag1 [87 (link)] 1/200 for 1 h at room temperature. αH3ac (rabbit, Millipore, 06–599B) 1/200, was incubated overnight. Appropriate secondary antibodies were used: phosphatase alkaline-conjugated goat anti-mouse or anti-rabbit (Sigma) along with the NBT and BCIP (Promega) detection system.

Free full text: Click here

Vanagas L., Muñoz D., Cristaldi C., Ganuza A., Nájera R., Bonardi M.C., Turowski V.R., Guzman F., Deng B., Kim K., Sullivan WJ J.r, & Angel S.O. (2023). Histone variant H2B.Z acetylation is necessary for maintenance of Toxoplasma gondii biological fitness. bioRxiv.

Publication Preprint 2023

αH3ac phosphatase alkaline-conjugated goat anti-mouse or anti-rabbit NBT and BCIP

Antibodies Buffer Goat Membrane Mouse Parasites Phosphatase Promega Proteins Pvdf Rabbit Sds page Western blot

Corresponding Organization : National University of General San Martín

Other organizations : Pontificia Universidad Católica de Valparaíso, University of Vermont, University of South Florida, Indiana University – Purdue University Indianapolis

Top 5 similar protocols

Variable analysis

independent variables

Number of cycles of rapid freezing/defreeze in hypotonic buffer (6 cycles)
Boiling with LB for 5 minutes

dependent variables

Expression of proteins (H2B.Z, H2A.Z, ROP5, ROP18, c-Myc, Sag1, H3ac)

control variables

Parasite count (0.5 to 1×10^7 parasites loaded per well)
SDS-PAGE gel (15%)
Protein transfer to PVDF membrane (1h at 100V)
Primary antibody concentrations (1/5000 for αrH2B.Z, αrH2A.Z, αrROP5, αrROP18; 1/2000 for α-c-Myc; 1/200 for αrSag1; 1/200 for αH3ac)
Incubation time for primary antibodies (1h at room temperature, except αH3ac incubated overnight)
Secondary antibodies (phosphatase alkaline-conjugated goat anti-mouse or anti-rabbit)
Detection system (NBT and BCIP)

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!