Proteins containing the V5 epitope were detected with a mouse monoclonal anti-V5 (Sigma, V8012) and HA-tagged proteins with a rabbit anti-HA (Sigma H6908). The rabbit polyclonals against eIF3i and eIF3h were sourced from Proteintech Group (11287-1-AP) and Sigma (AY50491), respectively. Other anti-eIF3 subunit antibodies used were; anti-eIF3a, Cell Signaling (#2538); anti-eIF3b, Santa Cruz (sc16377); anti-eIF3c, Sigma (E6408). A rabbit polyclonal against an N-terminal sequence of c-Myc was from Abcam (ab11917). Subunit-specific rabbit polyclonals against individual CCT subunits and an antibody to RPL7a have been characterized elsewhere (Roobol and Carden, 1999 (link)). α-tubulin was detected with the TAT mouse monoclonal (Woods et al., 1989 (link)) which was a kind gift from Prof. Keith Gull (University of Oxford, UK). β-actin was detected with the mouse monoclonal AC-15 (Sigma). For western blot detection of pulldown proteins a protein A-HRP conjugate (Millipore) was used, otherwise anti-rabbit, -mouse or -goat-HRP was used as secondary.
Anti eif3b
Manufactured by Santa Cruz Biotechnology
Sourced in Germany
Anti-eIF3b is a primary antibody that binds to the eIF3b (Eukaryotic Translation Initiation Factor 3 Subunit B) protein. eIF3b is a subunit of the eukaryotic translation initiation factor 3 (eIF3) complex, which plays a critical role in the initiation of protein synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Anti v5, by Merck Group (1 mentions)
Ac 15, by Merck Group (1 mentions)
Ab11917, by Abcam (1 mentions)
Anti eif3a, by Cell Signaling Technology (1 mentions)
Gammabind g sepharose, by GE Healthcare (1 mentions)
Anti g3bp1, by Santa Cruz Biotechnology (1 mentions)
Anti eif4a, by Santa Cruz Biotechnology (1 mentions)
Anti tiar, by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Avantor (1 mentions)
Las af software, by Leica (1 mentions)
Immunofluorescence microscope, by Olympus (1 mentions)
Mounting media, by Agilent Technologies (1 mentions)
Anti tia1, by Abcam (1 mentions)
Anti mouse cy2, by Jackson ImmunoResearch (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sodium meta arsenite, by Merck Group (1 mentions)
Elipse e800, by Nikon (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti eif3b
1
Antibody Detection for Protein Analysis
2
Immunoprecipitation of eIF3 Complexes
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eIF3 complexes were immunoprecipitated from WCEs, the preparation of which is described above, using GammaBind G Sepharose (GE Healthcare, cat # 17-0885-01) with either anti-eIF3b (Santa Cruz, cat # sc-16377) or anti-eIF3f (kind gift of Dr Hiroaki Imataka) primary antibodies. The detailed CoIP protocol was reported previously (39 (link)). To visualize western signals particularly from the eIF3f-CoIP, a protein-A linked to peroxidase (GE Healthcare, cat # NA9120) had to be used because of the rabbit origin of the eIF3f antibodies.
3
Antibody Detection in Cellular Assays
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Mouse anti-p24 was obtained from the National Institutes of Health AIDS Reference and Reagent Program. Anti-eIF4A (provided by Dr Simon Morley, University of Sussex, UK); anti-G3BP1 (provided by Dr Imed Gallouzi, McGill University, Canada); anti-TIAR (Santa Cruz Biotechnology); anti- eIF2α-P (Abcam); anti-eIF4GI (provided by Dr Nahum Sonenberg, McGill University, Canada); anti-eIF3b (Santa Cruz Biotechnology) and AlexaFluor fluorophore conjugates (Life Technologies).
4
Immunoprecipitation of eIF3b Protein Complexes
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Protein A/G-agarose (Pierce; catalog no. 20421) was preincubated with anti-eIF3b (Santa Cruz; catalog no. sc-16377) for 2 h while rotating at 4°C (around 2 μg of the antibody per 100 μl of the 50% protein A/G slurry in buffer A without protease inhibitors and Triton X-100). For the negative control, incubation was carried out without antibodies. Preincubated agarose beads were washed several times with buffer A, WCE diluted in buffer A was added (∼1 mg of total protein with the final concentration of Triton X-100 at ∼0.3%), and the mixture was incubated overnight at 4°C while rotating. Subsequent washing steps were performed with buffer A containing ∼0.3% of Triton X-100. Coimmunoprecipitated (co-IP) proteins were eluted in a denaturing loading buffer by boiling, and the eluate was analyzed by SDS-PAGE followed by Western blotting. Pretreatment of cells with a cross-linking agent (HCHO) had no visible effect on the final outcome and thus was not routinely used.
5
Immunofluorescence and Confocal Microscopy Protocol
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Immunofluorescence and confocal microscopy was carried out on cell lines grown on coverslips and fixed using 4% (w/v) paraformaldehyde (BDH Laboratory Supplies, Poole, UK) and permeabilised using 0.1% (v/v) Triton X-100 (Sigma Chemicals). Staining was carried out using anti-eIF3b (Santa Cruz, Heidelberg, Germany), anti-TIA-1 (Abcam, Cambridge, UK) and DAPI (Sigma Chemicals) was used as a nuclear stain. Coverslips were mounted onto microscopy slides using mounting media (Dako). Cells were imaged on the Olympus immunofluorescence microscope and captures using the Olympus 1 × 81 Camera (Olympus, Essex, UK). For confocal microscopy images were captures on a Leica microscope using the LAS AF software (Leica Microsystems, Milton Keynes, UK).
6
Stress Granule Induction and Visualization
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The day prior to the experiment, 105 U2OS cells were seeded onto 11 mm glass coverslips and allowed to attach overnight at 37 °C/5 % CO2 in DMEM containing 10 % FBS (Gibco). Cells were treated with 100 μM sodium (meta)arsenite (Sigma Aldrich) for 1 h to induce the formation of stress granules and then with 4 % paraformaldehyde solution at room temperature for 15 minutes followed by blocking and permeabilization with 5 % normal horse serum, 0.1 % digitonin in Tris-buffered saline. Staining was performed with anti-eIF3b (Santa Cruz), anti-SK1-Hedls (Santa Cruz), and patient sera for 1 h at room temperature. Secondary antibodies (anti-goat-Cy3, anti-mouse-Cy2, and anti-human-Cy5) were purchased from Jackson Laboratories and incubated at room temperature for 1 h. Conventional fluorescence microscopy was performed using a microscope (model Elipse E800, Nikon, Tokyo, Japan) with epifluorescence optics with a digital camera (model CCD-SPOT RT; Diagnostic Instruments, Sterling Heights, MI). Images were compiled using Adobe Photoshop software (CS6; Adobe Systems, San Jose, CA).
7
Immunofluorescence Staining of HeLa Cells
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HeLa cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min, washed three times with PBS. Nonspecific binding sites were blocked with 10% normal goat serum, 0.1% Triton X-100 for 1 h at room temperature. The cells were then stained for 2 h at room temperature with the primary binder / antibody. After that, the cells were washed three times with PBS containing 0.1% TritonX-100, following by staining for 1 h with the secondary antibody. Finally, cells were washed three times with PBS containing 0.1% Triton X-100, and mounted with ProLong Gold Antifade reagent with DAPI (Invitrogen). Alexa Fluor 488 or 647 conjugated F(ab')₂ fragment goat anti-human IgG, F(ab')2 fragment specific (Jackson ImmunoResearch) was used as the secondary antibody (1:500) when using sAB-K29 as the primary binder (2 μg/mL). Other primary antibodies are diluted as followed: Anti AuroraB (Fisher,1:500); Anti α-Tubulin (Genscript, 1:50-1:100); Anti ɑ-Tubulin (proteintech, 1:200); Anti c-Myc (Genscript, 0.5 μg/mL); Anti EIF3B (Santa Cruz, 1:100); Anti INCENP (Santa Cruz,1:100), Anti MKLP1(Santa Cruz, 1:100); Anti Proteasome 20S core subunits (Fisher, 1:250); Anti K48-Ub (Abcam, 1:200); Anti VCP (Santa Cruz,1:200); Anti G3BP1 (proteintech,1:500); Anti pTBK1 (cell signaling,1:50); Anti PLK1 (Santa Cruz,1:50). Images were collected by a Leica SP5 2photon laser scanning confocal microscope.
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