Transfected cells were analyzed by flow cytometry for the presence of VP1 or GFP. After trypsinization and harvesting in 2 ml of DPBS-20% FBS, the cells were fixed with 1% PFA (paraformalde-hyde) for 10 min at 4 °C followed by 10 min of permeabilization using triton 0.1%. Cell pellets were washed, and incubated with a 1:100 dilution of anti-VP1 antibody (Dako) for 30 min on ice. The primary antibody was removed by washing in DPBS-2% FBS, and cell pellets were incubated for an additional 30 min with a 1:500 dilution of anti-mouse Alexa Fluor 647 antibody (Life Technologies). After a final wash, the cells were resuspended in 500 μl of 1% PFA before analyzing with a BD FACSARIA III flow cytometer. The data were analyzed with FlowJo v10.7 (Tree Star Inc.).
Anti vp1 antibody
Manufactured by Agilent Technologies
The Anti-VP1 antibody is a laboratory reagent used for the detection and analysis of the VP1 protein. It is a highly specific and sensitive tool for researchers studying viral infections and related diseases. The antibody can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA), to identify and quantify the presence of the VP1 protein in biological samples.
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Lab products found in correlation
Facsaria 3, by BD (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Ezview red anti flag m2 affinity gel, by Merck Group (1 mentions)
Enterokinase, by Merck Group (1 mentions)
Ni nta beads, by GE Healthcare (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti vp1 antibody
1
Flow Cytometry Analysis of Transfected Cells
2
Immunoprecipitation of Viral Proteins
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Immunoprecipitation (IP) of VP1 was performed using anti-VP1 antibody (Dako, M706401-1) pre-incubated with Dynabeads Protein G (Invitrogen, 1003D) according to the manufacturer’s instructions. IP of Flag-tagged constructs was performed using EZviewTM Red ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich, F2426) according to the manufacturer’s instructions. In brief, HeLa cell lysates were incubated with anti-Flag M2 agarose beads at 4 °C overnight. After three washes, the bound proteins were eluted with 2× SDS sample buffer and then subjected to western blot analysis.
3
Recombinant Protein Expression and Purification
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The ABD gene was cloned into the previously prepared pET28a-VP1 plasmid through BamH I digestion sites. For evaluating dLNs accumulation of ABD-VP1 or VP1, the mCherry gene was cloned into the pET28a-ABD-VP1 or pET28a-VP1 plasmid. The inserted fragments were verified by DNA sequencing (Genewiz). Recombinant proteins were produced by the E.coli prokaryotic expression system and purified with His-beads. The specific expression and purification of those recombinant proteins were conducted as previously reported (22 (link)). Purified proteins were confirmed by western blot assays with the anti-VP1 antibody (Dako).
4
Recombinant VP1 Protein Expression
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Competent E. coli BL21 (DE3) cells were transformed with pERBC-VP1-RBN and pET–VP1, respectively. The cells were grown by continuous shaking at 37 °C, in LB medium containing 100 μg/ml ampicillin until OD≈0.8. Protein expression was then induced by adding IPTG to a final concentration of 1 mM and the incubation was extended for an additional 4 h at 37 °C. The cells were lysed by sonication and the target protein expressed as inclusion bodies. The cyclized and linear VP1 inclusion body proteins were purified and refolded as described previously41 . The solubilized VP1 from inclusion bodies were refolded by rapid dialysis. In order to get wild type form of linear VP1, the recombinant expressed VP1 protein from pET–VP1 was digested by enterokinase (Sigma) at 4 °C for overnight to remove the fusion tag at VP1 N terminus, then the mix was incubated with Ni-NTA beads (GE Healthcare) and collect the flow-through to obtained purified linear VP1 (L-VP1). Protein concentration was measured by Micro BCA Protein Assay Kit (Thermo) and protein purity was assessed by SDS-PAGE stained with Coomassie blue. Western blot was used for detection VP1 proteins by using anti-VP1 antibody (Dako).
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