Anti-Cullin1 and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Calbiochem (La Jolla, CA). Anti-EF1α, anti-Skp1, and anti-ubiquitin antibodies were obtained from Millipore (Billerica, MA). The Anti-flag tag monoclonal antibody was bought from Sigma chemicals (St. Louis, MO). HRP-conjugated anti-mouse and anti-rabbit IgG antibodies for immunoblot assays were obtained from Santa Cruz Biotech Inc. (Santa Cruz, CA). AlexaFluor488- and AlexaFluor594-conjuagted anti-mouse and anti-rabbit IgG antibodies were purchased from Molecular Probes (Invitrogen) for immunofluorescence assays. Human pooled sera were prepared from 10 scrub typhus patients (IFA titer≥1∶1280) and 10 healthy volunteers following institutional review board approval and the receipt of informed consent from all subjects.
Anti ef1α
Manufactured by Merck Group
Sourced in United States
Anti-EF1α is a laboratory reagent used to detect and quantify the presence of the protein Elongation Factor 1-alpha (EF1α) in biological samples. EF1α is a highly abundant protein involved in protein synthesis. Anti-EF1α is a specific antibody that can bind to and identify EF1α, allowing researchers to measure its levels in cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti parp, by Cell Signaling Technology (3 mentions)
Anti mcl 1, by Cell Signaling Technology (2 mentions)
Anti bcl xl, by Cell Signaling Technology (2 mentions)
Anti flag tag monoclonal antibody, by Merck Group (1 mentions)
Anti ubiquitin, by Merck Group (1 mentions)
Hrp conjugated anti mouse and anti rabbit igg antibodies, by Santa Cruz Biotechnology (1 mentions)
Anti bip grp78, by Santa Cruz Biotechnology (1 mentions)
Anti mcl 1, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Takara Bio (1 mentions)
Goat anti mouse igg hrp, by Southern Biotech (1 mentions)
Goat anti human igg hrp, by Southern Biotech (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Deae cellulose de52, by Cytiva (1 mentions)
Srx 101a x ray developer, by Konica Minolta (1 mentions)
Whatman, by Cytiva (1 mentions)
Anti xiap, by Cell Signaling Technology (1 mentions)
Anti cyclin b1, by Cell Signaling Technology (1 mentions)
Anti p p38 mapk, by Cell Signaling Technology (1 mentions)
Anti cyclin b1, by Abcam (1 mentions)
9 protocols using anti ef1α
1
Antibody Characterization for Immunoassays
2
Whole-Cell Lysate Preparation and Western Blot Analysis
3
Western Blot Analysis of LANA Protein
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Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes for western blot. Membranes were incubated with the primary antibody in PBS containing 0.05% Tween 20 (PBST) and 0.5% (weight/volume) non-fat dry milk at 4°C overnight followed by incubation with secondary antibody in PBST at room temperature for 1 hour. The following antibodies were used at the indicated dilutions: anti-GFP (632380, Clontech, 1/5000), anti-LANA immune serum (1/5000), anti-tubulin (66031-1-lg, Proteintech, 1/5000) anti-EF1α (05–235, Millipore, 1/5000). Secondary antibodies used were goat anti-mouse IgG HRP (1030–05, Southern Biotech, 1/5000) and goat anti-human IgG-HRP (2010–05, Southern Biotech, 1/5000).
For transfections, ~0.5 μg of GFP LANA 779–1162 or GFP LANA 933–1162 plasmid was used to transfect ~0.5x106 293 cells in one well of a 12 well plate, at which point cells were at ~70–80% confluence, using Lipofectamine 3000 (L3000015, Invitrogen) according to the manufacturer’s instructions. Transfected cells were harvested at 48h post transfection.
For transfections, ~0.5 μg of GFP LANA 779–1162 or GFP LANA 933–1162 plasmid was used to transfect ~0.5x106 293 cells in one well of a 12 well plate, at which point cells were at ~70–80% confluence, using Lipofectamine 3000 (L3000015, Invitrogen) according to the manufacturer’s instructions. Transfected cells were harvested at 48h post transfection.
4
Purification and Detection of Stumpy Trypanosomes
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Stumpy form trypanosomes were purified from blood using DEAE-cellulose DE52 (Whatman) anionic exchange columns [82 (link)] that were preincubated with PSG (44 mM NaCl, 57 mM Na2HPO4, 3 mM KH2PO4, 55 mM glucose, pH 7.8) warmed to 37°C. Western blotting and antibody concentrations were as described previously [35 (link)]. Anti-EF1α (Millipore) was used at a dilution of 1/7000. Proteins were detected using Enhanced Chemiluminescence reagents (Amersham) and a SRX-101A X-ray developer (Konica Minolta).
5
Western Blot Analysis of Cell Cycle Regulators
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Preparation of whole-cell lysates and Western blot analyses were performed as described [28 (link)]. The primary antibodies used were anti-CyclinB1 (1:1,000, rabbit polyclonal; Cell Signaling), anti-Cyclin B1 (1:1000, mouse monoclonal [V152]; Abcam), anti-CDK1 (1:1, 000, rabbit polyclonal; Cell Signaling), anti-pSer216-CDC25c (1:1,000, rabbit monoclonal; Cell Signaling), anti-CDC25c (1:1,000, rabbit monoclonal; Cell Signaling), anti-p-JNK (1:1,000, rabbit monoclonal; Cell Signaling), anti-JNK (1:1,000, rabbit polyclonal; Cell Signaling), anti-p-p38 MAPK (1:1,000, rabbit monoclonal; Cell Signaling), anti-p38 MAPK (1:1,000, rabbit monoclonal; Cell Signaling), anti-p-Bcl-xL (1:1,000, rabbit polyclonal; Thermo Scientific), anti-Bcl-xL (1:1,000, rabbit monoclonal; Cell Signaling), anti-Mcl1 (1:1,000, rabbit monoclonal; Cell Signaling), anti-XIAP (1:1,000, rabbit polyclonal; Cell Signaling) and anti-PARP (1:1,000, rabbit polyclonal; Cell Signaling). Blots were stripped and normalized by reprobing with anti-EF1α (1:2,000, mouse monoclonal; Millipore) and β-actin (1:20,000, mouse monoclonal; Sigma).
6
Western Blot Analysis of EF1α and GAPDH
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Cells were extracted in RIPA lysis buffer (ThermoFisher), supplemented with complete protease inhibitor and phosphatase inhibitor cocktail (ThermoFisher). After incubation on ice for 1 h, samples were centrifuged at 13,000g for 20 min at 4°C. Protein samples were separated in SDS-PAGE gel and then transferred onto a 0.2-mm polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membrane using wet transfer method at 90 V for 90 min. Primary antibodies anti-EF1α (Millipore #05-235) and anti-GAPDH (Cell Signaling Cat #2118S) were used with 1:1000 and 1:10,000 dilutions, respectively. Secondary antibodies HRP goat anti-rabbit (Vector Cat# PI-1000) and HRP horse anti-mouse (Vector Cat# PI-2000) were both diluted at 10,000. Proteins were detected using IVIS Lumina Imaging System.
7
Generation of Affinity-Purified Antibodies
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Generation of affinity-purified antibodies against γH2A (1:1000) from rabbit serum was previously described [84 (link)]. Commercial primary antibodies used here were: mouse anti-HA (1: 5000, Sigma), anti-EF1α (1: 40 000, Merck Millipore) and anti-BrdU clone B44 (1: 500, BD Bioscience). Commercial secondary antibodies used here were: goat anti-Rabbit IgG HRP-conjugated (ThermoFisher), goat anti-Rabbit IgG HRP-conjugated (ThermoFisher), goat anti-Rabbit IgG Alexa Fluor 488-conjugated (ThermoFisher), goat anti-Rabbit IgG IRDye 800CW-conjugated (Li-COR Biosciences) and goat anti-Mouse IgG IRDye 680CW-conjugated (Li-COR Biosciences).
8
Antibody Generation and Characterization
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Generation of affinity purified antibodies anti-LmRad9 (1: 3000), anti-LmHus1 (1: 2000), anti-Rpa1 (1: 1000), anti-γH2A (1: 5000) from rabbit serum, and chicken anti-Rad1 (1: 2000) from eggs, was previously described (17 (link),18 (link),26 (link)). Commercial primary antibodies used here were: mouse anti-Myc (1: 5000), anti-EF1α (1: 40 000) and anti-β-Tubulin-clone KMX1 (1: 1000) (Merck Millipore); mouse anti-BrdU (1: 500) (BD Bioscience); rabbit anti-H2A (1: 2000) (Santa Cruz). Commercial secondary antibodies used here were: goat anti-Rabbit IgG Alexa 488, anti-Rabbit IgG Alexa 594, anti-Mouse IgG Alexa 488 and anti-Mouse IgG Alexa 594 (Thermo Fisher).
9
Cell Lysis and Western Blot Analysis
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Cells were lysed in cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) and whole cell lysates were collected after centrifugation at 12,000 rpm for 15 min at 4°C [12 (link)]. For Western blotting analyses, the primary antibodies used were mouse monoclonal anti-MDA-7/IL-24 (1:2000; Gen Hunter Corporation, Nashville, TN, USA), anti-E1A (1:1000; EMD Millipore), anti-EF1α (1:5000; EMD Millipore), anti-β-actin (1:5000; Sigma-Aldrich), rabbit monoclonal anti-Bcl-xL (1:1000), anti-PARP (1:1000), anti-Bcl-2 (1:1000), anti-Mcl-1, rabbit polyclonal anti-phospho p-38 and anti-p38 (1:1000; Cell Signaling Technology). The secondary antibodies used were polyclonal goat anti-mouse IgG (1:1000; Dako, Carpinteria, CA, USA) and polyclonal swine anti-rabbit IgG (1:3000; Dako).
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