Ab126090

Manufactured by Abcam

Ab126090 is a lab equipment product. It is a general-purpose device used for various scientific applications in a laboratory setting.

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4 protocols using ab126090

Protein Expression Analysis in HeLa Cells

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RIPA protein extraction reagent (Beyotime, Shanghai, China) with PMSF (Roche, Basel, Switzerland) was used to lysed HeLa cells. 10% SDS-PAGE was used to separate the protein and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). 5% non-fat milk was used to block the membranes for 2 h. The membranes were probed with anti-GSPT1 (Abcam, ab126090), anti-WNT3A (Abcam, ab28472), anti-ICAM1 (Abcam, ab222736), anti-Vimentin (Abcam, ab8069), anti-E-cadherin (Abcam, ab53013), anti-p-β-catenin (Abcam, ab11350), β-catenin (Abcam,ab32572), anti-c-myc (Abcam, ab39688), anti-cyclin D1 (Abcam, ab226977), anti-GAPDH (Abcam, ab181602) antibodies overnight at 4˚C. Subsequently, membranes were incubated with a HRP-conjugated secondary antibody (CST, #7074) for 1 h at 37˚C. Finally, the blots were visualized by ECL (Thermo Fisher Scientific) and detected using a ChemiDoc XRS imaging system. Band densities were analyzed using Image J software (National Institute of Health, Bethesda, MD, USA). Relative protein levels were determined by normalizing the densitometry value of the proteins of interest to that of GAPDH.

Wu W., Guo L., Liang Z., Liu Y, & Yao Z. (2020). Lnc-SNHG16/miR-128 axis modulates malignant phenotype through WNT/β-catenin pathway in cervical cancer cells. Journal of Cancer, 11(8), 2201-2212.

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Protein Extraction and Analysis in Gastric Cancer

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A total cell protein extraction kit (KeyGen Biotechnology) was adopted for separating total proteins of gastric cancer tissues or cells. Next, following SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE), the transfer of total protein for every sample onto the polyvinylidene difluoride (PVDF) membranes was conducted. Next, 2% bovine serum albumin (KeyGen Biotechnology) was adopted to block PVDF membranes at room temperature for 2 h, and the overnight incubation of all these membranes was made at 4°C with the primary antibodies below: GSPT1 (1:1000; #ab126090, Abcam) and β‐actin (1:200; #ab115777, Abcam). After 2 h of secondary antibody incubation, the detection of all the bands was made with a chemiluminescence ECL kit (Beyotime Biotechnology), and all the bands were quantified with the ImageJ software (National Institutes of Health, Bethesda). The calculation of the expression of proteins was made on basis of the internal regulation β‐actin.

Liu R., Han X., Gao S., Chen Y, & Zhang J. (2023). Hsa_circ_0001944 enhanced GSPT1 expression via sponging miR‐498 to promote proliferation and invasion of gastric cancer. Journal of Clinical Laboratory Analysis, 37(2), e24810.

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Immunohistochemical Analysis of Gastric Cancer

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Embedded in paraffin, gastric cancer tissues were sliced into 4 mm sections and labeled with primary antibodies as follows: Ki67 (1:100; #ab92742, Abcam), GSPT1 (1:100; #ab126090, Abcam). Then, after the treatment with an immunohistochemical labeling kit (MaxVision Biotechnology), the slices were imaged via a light microscope (Olympus). According to the German immunohistochemical mark, the expression degrees and the staining intensity were evaluated.²⁹

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Western Blot Analysis of Protein Expression

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Cells
were seeded in 6-well plates (1 × 10⁶ cells per well).
After overnight incubation, the cells were treated with indicated
concentrations for specific timepoints. The harvested cells were spinned
down, washed with PBS, and lysed with 1× LDS loading buffer followed
by sonication and heated at 70 °C. The prepared samples were
loaded on NuPAGE 4–12% Bis–Tris protein gels and transferred
to the nitrocellulose membrane. After blocking the membrane with LI-COR
TBS blocking buffer and incubation in primary antibody overnight,
the corresponding protein signals were detected using IRDye secondary
antibodies and an Odyssey imaging system. Image Studio Lite Ver 5.2
was used for blot quantification. Rabbit anti-human eRF3/GSPT1 pAb
(ab126090, Abcam), mouse anti-human Ikaros mAb (sc-398265, Santa Cruz
Biotechnology), mouse anti-GAPDH mAb (sc-47724, Santa Cruz Biotechnology)
were used as primary antibodies. The IRDye 800CW Goat anti-Mouse IgG
Secondary Antibody and IRDye 680RD Goat anti-Rabbit IgG Secondary
Antibody were used as secondary antibodies.

Nishiguchi G., Keramatnia F., Min J., Chang Y., Jonchere B., Das S., Actis M., Price J., Chepyala D., Young B., McGowan K., Slavish P.J., Mayasundari A., Jarusiewicz J.A., Yang L., Li Y., Fu X., Garrett S.H., Papizan J.B., Kodali K., Peng J., Pruett Miller S.M., Roussel M.F., Mullighan C., Fischer M, & Rankovic Z. (2021). Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators. Journal of Medicinal Chemistry, 64(11), 7296-7311.

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