| Product Description | Aladdin's Positive Control Plasmid Pair for HA&Myc Co-IP contains two plasmids, pCMV-HA-p53 and pCMV-Myc-LTA, which can be used to express human p53 and LTA (large T antigen) proteins fused with with HA tag and Myc tag, respectively, at the N-terminus in mammalian cells. As p53 interacts strongly with LTA, this plasmid pair can be used as a positive control for Co- immunoprecipitation (Co-IP) of cotransfected cells or immunoprecipitation (IP) of separately transfected cells.IP and Co-IP are common experimental techniques to study protein or protein-protein interactions (PPIs) by using specific antibodies and resins capable of binding antibodies (e.g., Protein A/G Agarose or Protein A/G magnetic beads), or by employing directly the resins coupled with specific antibodies. The antigen and antibody complexes can be separated from the complicated protein solution by centrifugation or magnetic force, and then be analyzed by Western blot or mass spectrometry [1-2].The pCMV-HA-p53 plasmid is designed for the expression of p53. p53 (cellular tumor antigen p53) is 393 amino acids long and has a theoretical mass of 43.7 kDa. Its main functions include cell cycle regulation, promoting apoptosis, maintaining genomic stability and inhibiting tumor angiogenesis. p53 in wild type is an important oncogene [3].The pCMV-Myc-LTA plasmid is designed for the expression of LTA. LTA (SV40 large T antigen), also known as SV40gp6 large T antigen, is a hexameric protein consisting of 708 amino acids with a molecular weight of 81.6 kDa. When expressed alone, LTA induces the proliferation and transformation of quiescent cells and can induce tumor formation in experimental animals, providing a very useful model for studying DNA replication and cell transformation [4].Both plasmids contain CMV promoter which can efficiently initiate the expression of target proteins in cells, and both carry Ampicillin resistance and Neomycin resistance genes.Please refer to Figure 1A for the Co-IP of HA-p53 and Myc-LTA from cells co-transfected with Aladdin's Positive Control Plasmid Pair for HA&Myc Co-IP by magnetic beads, and Figure 1B for the Co-IP of HA-p53 and Myc-LTA by agarose gel.Figure 1. Co-Immunoprecipitation of HA-p53 and Myc-LTA from cells co-transfected with Aladdin's Positive Control Plasmid Pair for HA&Myc Co-IP . A. Myc-tag Protein IP Assay Kit with Magnetic Beads and HA-tag Protein IP Assay Kit with Magnetic Beads were used for Co-IP; B. Myc-tag Protein IP Assay Kit with Agarose Gel and HA-tag Protein IP Assay Kit with Agarose Gel were used for Co-IP. 293T cells (human embryonic kidney cells) were transfected with pCMV-HA-p53 and pCMV-Myc-LTA plasmids, respectively, or co-transfected for 36 hours, lysed using Aladdin's Western and IP Cell Lysis Buffer , and immunoprecipitated. Western blot imaging was performed by Imager 600 Chemiluminescent Imaging System . This figure is for reference only, which may vary due to different experimental conditions.The recommended sequencing primers are as follows
Precautions： This vector shall not be used for any commercial purposes without written permission from , and shall not be handed over to any individual or unit outside the laboratory where the order is placed.This plasmid pair has been validated by sequencing, immunoprecipitation and co-immunoprecipitation, but not validated by functional studies..This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： 1. If the D3045S package is ordered, please perform plasmid trasnformation in E. coli to amplify the plasmid prior to downstream applications. The extracted plasmids can be confirmed by enzyme digestion or DNA sequencing. 2. The D3045M package contains 100µg plasmids at a concentration of 0.25µg/µl, which can be used directly for cell transfection or enzyme digestion.
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