Ab86135

Manufactured by Abcam

Ab86135 is a primary antibody targeting protein X. It is suitable for use in various immunological techniques such as Western Blot, Immunohistochemistry, and Immunofluorescence. The antibody is produced in rabbit and is affinity-purified.

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Lab products found in correlation

4 protocols using ab86135

Cell Culture Maintenance and Genetic Manipulation

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HuH-7 and HEK293T cells were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen Strep, Gibco) in a 37°C humidified incubator with 5% CO₂. BHK-21 cells were propagated in RPMI Medium 1640 (Gibco) supplemented with 10% FBS and 1% Pen Strep at 37°C with 5% CO₂. C6/36 cells were maintained in RPMI media supplemented with 2% fetal calf serum (FCS) at 28°C. Tetracycline-inducible cell lines were generated using the Flp-In T-REx system (Thermo Fisher Scientific) according to manufacturer's protocol. After introduction of transgenes, HEK-293 Flp-In T-REx cells were grown in medium with 100 µg/mL of hygromycin B and 15 µg/mL of blasticidin. The following primary antibodies were utilized during western blotting, RNA immunoprecipitation, and focus formation assay techniques: rabbit G3BP1 antibody (A302-033; Bethyl Laboratories), rabbit G3BP2 antibody (ab86135; Abcam), rabbit CAPRIN1 antibody (15112-1-AP; Proteintech Group), rabbit DDX6 antibody (A300-460A; Bethyl Laboratories), rabbit QKI antibody (A300-183A; Bethyl Laboratories), mouse FLAG antibody (F3165; Sigma), mouse IgG antibody (12–371; Millipore) as isotype control, mouse pan-actin (MAS-11869; Thermo Fisher Scientific), and mouse pan flaviviral envelope 4G2 antibody.

Liao K.C., Chuo V., Ng W.C., Neo S.P., Pompon J., Gunaratne J., Ooi E.E, & Garcia-Blanco M.A. (2018). Identification and characterization of host proteins bound to dengue virus 3′ UTR reveal an antiviral role for quaking proteins. RNA, 24(6), 803-814.

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Immunostaining of Stress Granule Proteins

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Cells were grown on coverslips, washed with PBS and fixed for 20 min in 4% PFA. Cells were then permeabilized in 0.5% Triton X-100 for 3 min. After blocking, cells were immunostained for 1 h with a primary antibody, and after subsequent washes the cells were incubated for 1 h with fluorescently labeled secondary antibodies. Primary antibodies: goat anti-TIA-1 (Abcam, ab40693; 1:200), rabbit anti-eIF4B (Abcam, ab68474; 1:200), mouse anti-G3BP1 (Abcam, ab56574; 1:200), rabbit anti-G3BP2 (Abcam, ab86135; 1:1000), rabbit anti-α-tubulin (Abcam, ab4074; 1:400). Secondary antibodies: Alexa Fluor 488-labeled goat anti-mouse IgG and anti-rabbit IgG, Alexa Fluor 594-labeled goat anti-mouse IgG and anti-rabbit IgG (all from Abcam; 1:1000) and Alexa Fluor 647-labeled donkey anti-rabbit IgG (Life Technology; 1:1200). Nuclei were counterstained with Hoechst 33342 (Sigma), and coverslips were mounted in mounting medium (made in house).

Schwed-Gross A., Hamiel H., Faber G.P., Angel M., Ben-Yishay R., Benichou J.I., Ishay-Ronen D, & Shav-Tal Y. (2022). Glucocorticoids enhance chemotherapy-driven stress granule assembly and impair granule dynamics, leading to cell death. Journal of Cell Science, 135(14), jcs259629.

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Western Blot Analysis of Cell Signaling

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Cell lysate was collected using RIPA lysis buffer. The cell protein mass of each lysate was determined by BCA Protein Assay Reagent (Pierce, IL). Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to membranes (0.22 μm, Sigma) and incubated with primary antibodies at 4°C overnight. The primary antibodies are as follows: antibodies against VEGFA (1/1000; ab1316, Abcam), TGFβ1 (1/1000; ab215715, Abcam), Angiopoietin-1 (1/20000; ab183701, Abcam), ZO-1 (1/1000; ab216880, Abcam), Occludin (1/1000; ab216327, Abcam), Claudin-5 (1/1000; ab131259, Abcam), G3BP2 (1/2000; ab86135, Abcam), p-p38 (1/1000; ab195049, Abcam), p38 (1/2000; ab170099, Abcam), p-p53 (1/2000; ab33889, Abcam), p53 (1/1000; ab26, Abcam), and GAPDH (1/500; ab8245, Abcam). Next, the membranes were incubated with HRP-conjugated secondary antibody IgG (1/2000; ab7090, Abcam) at room temperature for 2 h. GAPDH antibody served as a negative control. At last, the protein bands were visualized by an ECL Western Blotting Substrate Kit (ab65623, Abcam) and quantified by the ImageJ software.

Zhao H, & He Y. (2021). MiR-124-3p Suppresses the Dysfunction of High Glucose-Stimulated Endothelial Cells by Targeting G3BP2. Frontiers in Genetics, 12, 723625.

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Antibodies for Western Blot and Immunofluorescence

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The antibodies used for Western blot analysis and immunofluorescence included mouse anti-HA (1:2000 for Western blot and 1:800 for immunofluorescence; H3663) from Millipore-Sigma, mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000; g9295) from Millipore-Sigma, mouse anti-G3BP1 (1:500 for Western blot and 1:200 for immunofluorescence; sc-365338) from Santa Cruz Biotechnology, rabbit anti-G3BP2 (1:2000; ab86135) from Abcam, rabbit anti-TIA1 (1:300; 12133-2-AP) from Proteintech, rabbit anti-CAPRIN1 (1:2000; 15112-1-AP) from Proteintech, rabbit anti-UBAP2L (1:4000; ab70319) from Abcam, mouse anti-actin (1:10,000; A5441) from Millipore-Sigma, mouse anti-tubulin (1:1000; T8328) from Millipore-Sigma, mouse anti-Satb2 (1:100; ab51502) from Abcam, rat anti-Ctip2 (1:200; ab18465) from Abcam, rabbit anti-Tbr1 (1:200; ab31940) from Abcam, rabbit anti-Pax6 (1:200; 901301) from BioLegend, and rabbit anti-Tbr2 (1:200; ab23345) from Abcam.

Jia X., Zhang S., Tan S., Du B., He M., Qin H., Chen J., Duan X., Luo J., Chen F., Ouyang L., Wang J., Chen G., Yu B., Zhang G., Zhang Z., Lyu Y., Huang Y., Jiao J., Chen J.Y., Swoboda K.J., Agolini E., Novelli A., Leoni C., Zampino G., Cappuccio G., Brunetti-Pierri N., Gerard B., Ginglinger E., Richer J., McMillan H., White-Brown A., Hoekzema K., Bernier R.A., Kurtz-Nelson E.C., Earl R.K., Meddens C., Alders M., Fuchs M., Caumes R., Brunelle P., Smol T., Kuehl R., Day-Salvatore D.L., Monaghan K.G., Morrow M.M., Eichler E.E., Hu Z., Yuan L., Tan J., Xia K., Shen Y, & Guo H. (2022). De novo variants in genes regulating stress granule assembly associate with neurodevelopmental disorders. Science Advances, 8(33), eabo7112.

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