A total of 5 × 105 HeLa cells were plated on glass coverslips in a 24-well plate and fixed with 4% PFA (Sigma) in PBS for 20 min at RT, washed in PBS, and stored at 4 °C. Cells were permeabilized with 0.1% Triton-X100 (Sigma) in PBS for 7 min, then blocked with 0.5% bovine serum albumin (Fisher) in PBS for 1 h. Coverslips were then incubated with 200 μl of primary antibody solution for 1 h at RT and washed 3 times with PBS before incubation with secondary antibody solution containing 0.1 μg/ml DAPI solution (Sigma) for 1 h at RT. After three washes with PBS, coverslips were mounted to microscope slides with 7 μl Mowiol 488 (Sigma). Confocal microscopy was performed on a Ti-Eclipse A1MP Multiphoton Confocal Microscope (Nikon) using the Nikon acquisition software NIS-Elements AR. Primary antibody dilution: Rabbit anti-G3BP1 (1:600, Sigma), Goat anti-eIF3B (1:600, Santa Cruz), mouse anti-puromycin (1:5, http://dshb.biology.uiowa.edu/PMY-2A4 ), Rabbit anti-UBAP2L (1:600, Santa Cruz). Secondary antibodies were all purchased from Invitrogen: Chicken anti-mouse Alexa 488, donkey anti-goat Alexa 555, and goat anti-rabbit 488.
Goat anti eif3b
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Goat anti-eIF3B is an antibody that recognizes the eIF3B subunit of the eukaryotic translation initiation factor 3 (eIF3) complex. eIF3B is a key component of the eIF3 complex, which plays a crucial role in the initiation of protein synthesis by facilitating the recruitment of the 40S ribosomal subunit to the mRNA.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti g3bp1, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Dapi solution, by Merck Group (1 mentions)
Donkey anti goat alexa 555, by Thermo Fisher Scientific (1 mentions)
Mowiol 4 88, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Goat anti rabbit 488, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Rabbit anti eif2α, by Cell Signaling Technology (1 mentions)
Mouse anti puromycin, by Merck Group (1 mentions)
Rabbit anti 4e bp1, by Cell Signaling Technology (1 mentions)
Rat anti tubulin, by Abcam (1 mentions)
Mouse anti g3bp1, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti eif4g, by Cell Signaling Technology (1 mentions)
Rabbit anti caprin 1, by Proteintech (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Rockland Immunochemicals (1 mentions)
Rabbit anti phospho eif2α ser51, by Cell Signaling Technology (1 mentions)
Mouse anti eif2α, by Cell Signaling Technology (1 mentions)
Mouse anti actin, by Abcam (1 mentions)
4 protocols using goat anti eif3b
1
Immunofluorescent Profiling of HeLa Cells
2
Molecular Profiling of Translation Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary antibodies were used in this study: mouse anti-G3BP1 (Santa Cruz, sc-81940, Santa Cruz, CA, USA), goat anti-eIF4AI (Santa Cruz, sc-14211), mouse anti-eIF4E (P-2, Santa Cruz, sc-9976), rabbit anti-eIF4G (Cell Signaling #2498), goat anti-eIF3B (Santa Cruz, sc-16377), rabbit anti-Caprin1 (Proteintech, 15112-1-AP, Chicago, IL, USA), rabbit anti-phospho(S51)-eIF2α (Cell Signaling #9721), rabbit anti-eIF2α (Cell Signaling #9722, Danvers, MA, USA), rabbit anti-4E-BP1 (Cell Signaling, #53H11), rabbit anti-phospho-4E-BP1(Thr37/46) (Cell Signaling, #236B4), rabbit anti-AMPKalpha (Cell Signaling, #2603), rabbit anti-phospho-AMPKalpha (Cell Signaling, #2535), rat anti-tubulin (Abcam, #ab6160, Cambridge, UK), rabbit anti-Trx1 (FL-105, Santa Cruz sc-20146) (WB), rabbit anti-Trx1 (Cell Signaling #2429) (IF) and mouse anti-puromycin (Millipore MABE343).
3
Antibody Validation for Immunodetection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit anti-Staufen1 antiserum generated to the full-length recombinant protein was produced and purified at the McGill University Cell Imaging and Analysis Network, and was used at 1:1000 for western blotting; mouse anti-p24 (clone 183-H12-5C) (NIH AIDS Reference and Reagent Program) was used to identify Gag at 1:400 for indirect immunofluorescence and at 1:10,000 for western blotting; and goat anti-TIAR1 (Santa-Cruz Biotechnology) was used for indirect immunofluorescence microscopy at 1:500; rabbit anti-phospho-eIF2α (Ser51) (Cinti et al. 2016 (link)) (Cell Signaling Technology) was used for indirect immunofluorescence microscopy at 1:200 and for western blotting at 1:1000; goat anti-eIF3b (Santa-Cruz Biotechnology) 1:250, as described by Valiente-Echeverría et al. (2014) (link); mouse anti-eIF2α (Cell Signaling Technology) and mouse anti-actin (Abcam) were used for western blotting at 1:1000 and at 1:10,000, respectively. Horseradish peroxidase-conjugated secondary antibodies were purchased from Rockland Immunochemicals, AlexaFluor secondary antibodies were purchased from Life Technologies, and were used as recommended by the manufacturers.
4
Western Blot Analysis of Stress Granule Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated at 2 × 105 in 6-well plates. At the indicated times, cells were lysed in 150 μl of 1× Gel Loading Buffer (New England Biolabs), sonicated and boiled 5 min at 95 °C. Cell lysates were separated by SDS-PAGE, and protein was transferred to nitrocellulose or polyvinylidene difluoride membranes. These were then probed with the following primary antibodies: rabbit anti-eIF2a (1:1,000, Cell Signalling), mouse anti-P-eIF2α (1:1,000, Cell Signalling), rabbit anti-G3BP1 (1:2,000, Sigma), rabbit anti-Caprin1 (1:1,000, Bethyl Laboratories), goat anti-eIF3B (1:2,000, Santa Cruz), mouse anti-GAPDH (1:20,000, Invitrogen), followed by incubation with the appropriate peroxidase-labeled secondary antibodies (Dako) and chemiluminescence development using the Clarity Western ECL Substrate (Bio-Rad). The results were acquired using the VILBER imaging system.
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!