Goat anti eif3η

Manufactured by Santa Cruz Biotechnology

Goat anti-eIF3η is a laboratory reagent used to detect the eIF3η subunit of the eukaryotic translation initiation factor 3 (eIF3) complex. eIF3η is a component of the eIF3 complex, which plays a crucial role in the initiation of protein synthesis in eukaryotic cells. This antibody can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of the eIF3η protein.

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Lab products found in correlation

Goat anti tia1, by Santa Cruz Biotechnology (2 mentions) Puromycin, by Thermo Fisher Scientific (1 mentions) Sodium arsenite, by Merck Group (1 mentions) Mouse anti gapdh, by Abcam (1 mentions) Pkr inhibitor c16, by Merck Group (1 mentions) Isrib, by Merck Group (1 mentions) Rabbit anti actin, by Merck Group (1 mentions) Mouse anti g3bp1, by Abcam (1 mentions) Rabbit anti calnexin, by Abcam (1 mentions) Poly 1 c, by Merck Group (1 mentions) Mouse anti puromycin, by Kerafast (1 mentions) Rabbit anti pabp, by Abcam (1 mentions) Rabbit anti tiar, by Cell Signaling Technology (1 mentions) Alexa fluor 488 tyramide superboost kit, by Thermo Fisher Scientific (1 mentions) Rabbit anti tdp 43, by Proteintech (1 mentions) Rabbit anti ha, by Cell Signaling Technology (1 mentions) Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions) Mouse anti flag, by Merck Group (1 mentions) Chicken anti gfp, by Abcam (1 mentions) Mouse anti g3bp1, by BD (1 mentions)

3 protocols using goat anti eif3η

Molecular Insights into Viral Stress Response

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Sodium arsenite (Sigma-Aldrich) was added to the cells at a concentration of 250 μM for 20 min prior to fixation or cell lysate collection. The PKR inhibitor C16 (Sigma-Aldrich) was added to infected cells at a concentration of 1 μM at 1 hpi, and cell lysates were collected at 12 hpi. The ISR inhibitor ISRIB (Sigma-Aldrich) was added at 1 hpi at 0.5 μM. Puromycin (Life Technologies) was added to cells at a concentration of 10 μg/ml at indicated times prior to cell lysate collection. Poly(I⋅C) (Sigma-Aldrich, catalog no. P1530) is a dsRNA analogue and was used as a positive control for immune activation. Poly(I⋅C) was used at a concentration of 20 μg/ml and added to the cell culture medium in combination with Lipofectamine 2000. MNV ORF1 plasmids were described and used previously (22 (link)).
Goat anti-eIF3η, goat anti-G3BP1, and goat anti-TIA-1 were all purchased from Santa Cruz Biotech. Rabbit anti-eIF2α was purchased from Invitrogen; Rabbit anti-actin from Sigma-Aldrich; Mouse anti-Puromycin was obtained from Kerafast, Inc. Mouse anti-G3BP1, mouse anti-GAPDH, rabbit anti-His, and rabbit anti-calnexin were obtained from Abcam, and rabbit anti-p-eIF2α (S52) and Alexa Fluor-conjugated species-specific IgG were purchased from Life Technologies. Rabbit anti-NS7 and rabbit anti-NS5 were manufactured and produced by Invitrogen.

Fritzlar S., Aktepe T.E., Chao Y.W., Kenney N.D., McAllaster M.R., Wilen C.B., White P.A, & Mackenzie J.M. (2019). Mouse Norovirus Infection Arrests Host Cell Translation Uncoupled from the Stress Granule-PKR-eIF2α Axis. mBio, 10(3), e00960-19.

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Immunofluorescence Analysis of Stress Granules

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Cells growing on glass-bottom dishes (ibidi; 81156) were fixed in 4% PFA for 5 min at room temperature, permeabilized with 0.1% TBST, blocked in 3% BSA, and then incubated with one of the following primary antibodies at 4°C overnigh t: mouse anti-FLAG (also recognizes the “DDK” tag) (Sigma Aldrich; F3165), mouse anti-G3BP1 (BD Biosciences; 611126), chicken anti-GFP (Abcam; ab13970), goat anti-TIA1 (Santa Cruz Biotechnology; sc-1751), rabbit anti-TIAR (Cell Signaling; 8509S), goat anti-eIF3η (Santa Cruz Biotechnology; sc-16377), rabbit anti–TDP-43 (Proteintech; 12892–1-AP), rabbit anti-PABP (Abcam; ab21060), rabbit anti-HA (Cell Signaling; 3724). Cells were then incubated with secondary antibodies conjugated to Alexa-488, −555 or −647 (Invitrogen), and mounted with ProLong Gold Antifade Reagent with DAPI (Invitrogen; P36931). To detect endogenous ULK1 and ULK2, the signal was amplified using Alexa Fluor 488 Tyramide SuperBoost Kit (Thermo Fisher Scientific; B40941) per the manufacturer’s instructions. Quantification of cells with stress granules was performed as previously described (Buchan et al., 2013 (link)).

Wang B., Maxwell B.A., Joo J.H., Gwon Y., Messing J., Mishra A., Shaw T.I., Ward A.L., Quan H., Sakurada S.M., Pruett-Miller S.M., Bertorini T., Vogel P., Kim H.J., Peng J., Taylor J.P, & Kundu M. (2019). ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97. Molecular cell, 74(4), 742-757.e8.

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Subcellular Localization of eIF3η and G3BP1

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U2OS cells (WT or G3BP1/2 KO cells) were seeded with a Thermo MultiDrop dispenser into a Greiner μClear 384 well plate (Greiner Cat #781092) and treated as described in the previous sections. Cells were fixed with 3.7% formaldehyde diluted in PBS for 10 min, washed with PBS and subsequently permeabilized with 0.2% Triton X-100 in PBS for 10 min. Cells were then blocked with 3% BSA in PBS for 1 hr and incubated overnight with primary antibodies (goat anti-eIF3η (Santa Cruz Biotechnology, sc-16377) diluted 1:2000 and rabbit anti-G3BP1 (Thermo Fisher, PA5-29455) diluted 1:500 in blocking solution). After washing with PBS, cells were incubated for 1 h with secondary antibodies (donkey anti-goat Alexa-647 and donkey anti-rabbit Alexa 488 diluted 1:1000 in blocking solution). The cytoplasm was stained with CellMaskBlue (Invitrogen) and the nuclei were stained with Hoechst (Invitrogen). Images were acquired on an automated confocal microscope Yokogawa cv7000 with a 40x 0.95 NA air lens.

Guillén-Boixet J., Kopach A., Holehouse A.S., Wittmann S., Jahnel M., Schlüßler R., Kim K., Trussina I.R., Wang J., Mateju D., Poser I., Maharana S., Ruer-Gruß M., Richter D., Zhang X., Chang Y.T., Guck J., Honigmann A., Mahamid J., Hyman A.A., Pappu R.V., Alberti S, & Franzmann T.M. (2020). RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation. Cell, 181(2), 346-361.e17.

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