T98G cells were transiently transfected with pEF6 vectors encoding V5-tagged wild-type or mutant LIN54 alleles and pcDNA3.1 vectors encoding haemagglutinin (HA)-tagged wild-type p130. Cells were extracted using EBC lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, protease and phosphatase inhibitor cocktails), immunoprecipitated using anti-V5 antibody (AbD Serotec) and subjected to western blot analysis using rabbit antibodies specific to human LIN9 and LIN37, made in collaboration with Bethyl, anti-Myb (sc-724) (Santa-Cruz Biotech), rabbit anti-V5 (Bethyl Inc.) or mouse monoclonal anti-HA (12CA5), rabbit anti-HA (sc-805) (Santa-Cruz Biotech)2 (link). The LIN9, LIN37, Myb and V5 antibodies were used at 1:1,000 dilution. The HA antibody was used at 1:15,000 dilution. Full western blottings are shown in Supplementary Fig. 9 .
Anti v5 antibody
Manufactured by Bio-Rad
Sourced in United Kingdom, United States
The Anti-V5 antibody is a laboratory reagent used for the detection and purification of proteins tagged with the V5 epitope. The antibody specifically binds to the V5 tag, allowing for the identification and isolation of V5-tagged proteins from biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Sp6 rna polymerase, by Promega (3 mentions)
Protein g dynal beads, by Thermo Fisher Scientific (2 mentions)
Anti flag antibody, by Merck Group (2 mentions)
Protein a sepharose cl 4b, by GE Healthcare (2 mentions)
Dinucleotide 4 thio upg, by Horizon Discovery (2 mentions)
Rnasin and t4 rna ligase 2, by Qiagen (2 mentions)
Rnase p1, by Merck Group (2 mentions)
Protein g sepharose beads, by Merck Group (2 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
Streptavidin sepharose, by GE Healthcare (1 mentions)
Rnasin, by Promega (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Irdye conjugated secondary antibody, by LI COR (1 mentions)
Anti gfp antibody, by Roche (1 mentions)
Hrp coupled secondary antibody, by Agilent Technologies (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Rnase h, by Promega (1 mentions)
Proteinase k, by MDBio (1 mentions)
Igg sepharose, by GE Healthcare (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
14 protocols using anti v5 antibody
1
LIN54 Protein Interaction Assay
2
Antibody Generation for Spliceosomal Proteins
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The anti-V5 antibody was purchased from Serotec Inc. The 8G5F antibody against hemagglutinin (HA) was produced by immunizing mice with a keyhole limpet hemocyanin-conjugated HA peptide (T.Y. Tsao and S.-C. Cheng, unpublished results). Antibodies against Prp19, Ntc90, Ntc85, Ntc77, Ntc30, Ntc25, Ntc20, Yju2 and Smd1 were produced by immunizing rabbits with corresponding recombinant proteins. Anti-Snu114 antibody was raised against amino acid residues 1–129 of Snu114 fused with GST, anti-Prp8 antibody against amino acid residues 1–115 of Prp8 and anti-Prp9 antibody against amino acid residues 1–161 of Prp9. RNasin and SP6 RNA polymerase were from Promega. Streptavidin Sepharose and protein A-Sepharose (PAS) were purchased from GE Healthcare Inc.
3
Isolation of Tagged Neuronal Nuclei
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Antibody-adsorbed Dynal Protein-G beads (Invitrogen: 100-03D) were generated as described previously57 (link). Briefly, 300 µl of beads were bound to 1µg of anti-GFP (Invitrogen: G10362), anti-FLAG (Sigma: F7425) or anti-V5 antibody (Abdserotec: MCA1360) in 1xPBS-0.01%Tween-20.
10XUAS-unc84-smFP_FLAG and 10XUAS-unc84-smFP_V5 reporter lines were generated by phiC31-mediated transgenesis58 (link). Tdc-GAL459 (link) was used to drive expression of both tags in octopaminergic neurons and nuclei were purified by the INTACT procedure57 (link). Approximately 200 frozen heads were added to 20 ml homogenization buffer (10 mM β-glycerophosphate pH 7.0, 2 mM MgCl2, 0.5% NP-40) and passed over a Yamato continuous flow homogenizer, set at 100 rpm, 6 times. The homogenate was then successively filtered through first a 20 µm and then a 10 µm nylon filter (Partec: 040042325, 0400422314). 60 µl of Dynal Protein-G magnetic beads adsorbed to anti-GFP, anti-FLAG or anti-V5 antibody were then added to the filtered homogenate, which was subjected to constant agitation at 4 °C for 30 minutes. Bead-bound nuclei were captured on a magnet, washed 3x with homogenization buffer, filtered through a 10 µm nylon filter and analyzed by both light and fluorescence microscopy.
10XUAS-unc84-smFP_FLAG and 10XUAS-unc84-smFP_V5 reporter lines were generated by phiC31-mediated transgenesis58 (link). Tdc-GAL459 (link) was used to drive expression of both tags in octopaminergic neurons and nuclei were purified by the INTACT procedure57 (link). Approximately 200 frozen heads were added to 20 ml homogenization buffer (10 mM β-glycerophosphate pH 7.0, 2 mM MgCl2, 0.5% NP-40) and passed over a Yamato continuous flow homogenizer, set at 100 rpm, 6 times. The homogenate was then successively filtered through first a 20 µm and then a 10 µm nylon filter (Partec: 040042325, 0400422314). 60 µl of Dynal Protein-G magnetic beads adsorbed to anti-GFP, anti-FLAG or anti-V5 antibody were then added to the filtered homogenate, which was subjected to constant agitation at 4 °C for 30 minutes. Bead-bound nuclei were captured on a magnet, washed 3x with homogenization buffer, filtered through a 10 µm nylon filter and analyzed by both light and fluorescence microscopy.
4
Quantifying GFP-Cps1 Sorting and Visualizing Vps36 Variants
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For quantification of GFP-Cps1 sorting into vacuole and for visualizing expression of the various tagged-Vps36, yeast cell extracts were obtained from strains grown to logarithmic phase. Cells were pelleted, resuspended in SDS–PAGE sample buffer (2% SDS, 0.1 M Tris, pH 6.8, 10% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol), lysed using glass beads, and boiled for 5 min at 95°C. For Western blotting, monoclonal antibodies against V5 and GFP (anti-V5 antibody from AbD Serotec [Kidlington, UK] and anti-GFP antibody from Roche Diagnostics [Basel, Switzerland]) were used at 1:2500 dilutions. The anti-Snf7 antibody used was previously described (Babst et al., 1998 (link)). IRDye-conjugated secondary antibody were purchased from LI-COR Biosciences (Lincoln, NE), and Western blots were imaged using the accompanying Odyssey Imaging System.
5
Immunoblotting with Commercial Antibodies
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The antibodies DO-1 (p53), PC10 (anti-proliferating nuclear cell antigen; PCNA) and 9E10 (Myc) were purchased from Santa Cruz. The anti-V5 antibody was obtained from Serotec and the anti-Flag antibody was from Sigma. HRP-coupled secondary antibodies were purchased from Dako.
6
Monoclonal and Polyclonal Antibody Production
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Anti-HA antibody 8G5F is a monoclonal antibody produced by immunizing mice with a keyhole limpet hemocyanin-conjugated HA peptide. The anti-V5 antibody was purchased from Serotec Inc. Anti-Lea1, anti-Prp5, anti-Msl5 and anti-Prp8 are polyclonal antibodies produced by immunizing rabbits with full-length recombinant protein (Lea1) or a segment of the corresponding proteins (amino acid residues 510–480 for Prp5, 273–412 for Msl5 and 1–115 for Prp8) expressed in Escherichia coli. Protein A-Sepharose CL-4B was purchased from GE Healthcare, SuperScript III from Invitrogen, proteinase K from MD Bio Inc. and RNase H from Promega. Dinucleotide 4-thio-UpG was purchased from Dharmacon. SP6 RNA polymerase was purchased from Promega, RNase P1 from Sigma, and RNasin and T4 RNA ligase 2 were from Enzymatics.
7
Antibody Production and Purification
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Anti-V5 antibody was purchased from Serotec Inc. Anti-HA antibody was produced by immunizing mice with a keyhole limpet hemocyanin (KLH)-conjugated HA peptide (unpublished). Anti-Prp16, anti-Prp22, anti-Prp8, anti-Ntc20, anti-Yju2, anti-Cwc2 and anti-Cwc25 polyclonal antibodies were produced by immunizing rabbits with fragments or full-length recombinant Prp16 (1-298), Prp22 (1-484), Prp8 (1-115), Ntc20, Yju2, Cwc2 and Cwc25 proteins, respectively. Dinucleotide 4-thio-UpG was purchased from Dharmacon. SP6 RNA polymerase was purchased from Promega, RNase P1 from Sigma, and RNasin and T4 RNA ligase 2 from Enzymatics. Protein A-Sepharose CL-4B and IgG Sepharose were purchased from GE Healthcare. Complete, EDTA-free Protease Inhibitor Cocktail was purchased from Roche. TEV protease with a mutation (Ser219Asn) at the internal self-cleavage site was a kind gift of Hung-Ta Chen (Institute of Molecular Biology, Academia Sinica).
8
Evaluation of ErbB2 Homodimer Formation
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Homodimer of ErbB2 was aslo evaluate in a coimmunoprecipitation assay, as describe [11 (link), 16 (link), 17 (link)]. Transfected cells were lysed in 1 ml RPMI lysis buffer (1% v/v DevelopTriton X-100, 1% w/v CHAPS, 10 mM HEPES (pH 7.2), in RPMI medium containing 0.2 mM PMSF, 10 ug/ml leupeptin, 10 U/ml aprotinin, and 1 mM Na3VO4). Homodimers were immunoprecipitated from 500 ul of lysate using mouse anti-V5 antibody(AbD Serotec) covalently coupled to agarose (Pierce ultralink) at 4°C for 2 h. Complexes were washed twice in lysis buffer and resuspended in SDS sample buffer and boiled. Samples were separated on a 4%–12% polyacrylamide gel (Novex) and electro-blotted onto nitrocellulose membranes. Blots were blocked in 10% BSA/TBST and probed with an anti-Flag antibody(F3165, Sigma) to detect ErbB2 followed by a peroxidase-conjugated anti-mouse secondary antibody (Amersham) To verify and normalize for expression of transfected constructs between experimental conditions, 50ug of cell lysate was checked by western blotting with anti-V5 antibody (AbD Serotec) or anti-Flag antibody(Sigma). Anti-mouse HRP-conjugated secondary antibody was used for visualization by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
9
Isolation of Tagged Neuronal Nuclei
10
Characterization of Recombinant EHDV VP7 Protein
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Recombinant VP7 purified from Sf9 cell pellet and supernatant were checked for purity by SDS-PAGE and characterized by Western blotting using the anti-VP7 mAb, clone 4G11 (IZSLER) raised against semipurified EHDV field strain serotype 7 obtained from Kimron Institute (Israel) and the anti-V5 antibody (Thermo Fisher Scientific, Cat#R961-25, RRID: AB_2556565) both conjugated with HRP. Uninfected Sf9 cells were used as negative control.
Briefly, the same quantity of purified protein derived from cell pellet and supernatant was denatured for 10 min at 70°C, as suggested by Thermo Fisher Scientific instructions, separated on NuPAGE Novex 4–12% Bis-Tris Gels (Thermo Fisher Scientific, #CatNP0321BOX) and stained with Biosafe Coomassie G-250 stain (Bio-Rad, #Cat 1610786).
Recombinant VP7 samples fractionated by electrophoresis were also transferred onto nitrocellulose membranes and tested for immunoreaction, incubating the membranes with the anti-VP7 mAb clone 4G11 (IZSLER) and the anti-V5 antibody at 1 : 30000 dilution and 1 : 10000 dilution, respectively, both conjugated with HRP, at 4°C overnight. After rinsing membranes with PBST, the Amersham ECL Select Western Blotting Detection Reagent (Cytiva, Cat# RPN2235) was used to acquire images of EHDV recVP7.
Briefly, the same quantity of purified protein derived from cell pellet and supernatant was denatured for 10 min at 70°C, as suggested by Thermo Fisher Scientific instructions, separated on NuPAGE Novex 4–12% Bis-Tris Gels (Thermo Fisher Scientific, #CatNP0321BOX) and stained with Biosafe Coomassie G-250 stain (Bio-Rad, #Cat 1610786).
Recombinant VP7 samples fractionated by electrophoresis were also transferred onto nitrocellulose membranes and tested for immunoreaction, incubating the membranes with the anti-VP7 mAb clone 4G11 (IZSLER) and the anti-V5 antibody at 1 : 30000 dilution and 1 : 10000 dilution, respectively, both conjugated with HRP, at 4°C overnight. After rinsing membranes with PBST, the Amersham ECL Select Western Blotting Detection Reagent (Cytiva, Cat# RPN2235) was used to acquire images of EHDV recVP7.
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