IP format Protein immunoprecipitation protocol Use protein IP to enrich a target protein from lysate before Western blot, mass spectrometry, activity testing, or other downstream analysis. The main goal is to pull down the target while minimizing non-specific bead and antibody background. Use this workflow when: Target enrichment before Western blot, confirmation of target pull-down, protein complex cleanup, or low-abundance protein detection. Optimize first: Antibody suitability for IP, bead type, lysis buffer, wash stringency, and elution method. Prepare lysate Lyse cells or tissue under cold, non-denaturing conditions when native antibody recognition is required. Clarify lysate thoroughly before adding antibody or beads. Use fresh protease inhibitors and phosphatase inhibitors when modification state matters.Save an input aliquot before the IP. Pre-clear the sample Incubate lysate with control beads to reduce proteins that bind beads non-specifically. Pre-clearing is especially useful with sticky lysates, tissue extracts, or high-abundance proteins.Keep the pre-cleared lysate as the starting material for the antibody pull-down. Bind antibody and target Incubate lysate with the IP antibody under conditions that preserve the target epitope. Use an antibody validated or suitable for immunoprecipitation when available.Run an IgG control antibody in parallel. Capture with beads and wash Add Protein A, Protein G, or compatible beads, then wash enough to reduce background without losing target. Choose bead type based on antibody species and isotype.Increase wash stringency only after confirming target recovery. Elute and analyze Elute bound protein using a method compatible with the downstream assay. For Western blot, account for antibody heavy chain near 50 kDa and light chain near 25 kDa.Use light-chain-specific, conformation-specific, or directly conjugated detection if antibody chains interfere.
Cart (0)
![5-Methylcytosine (5-mC) Monoclonal Antibody [33D3]](images/a1014data1.gif "5-Methylcytosine (5-mC) Monoclonal Antibody [33D3]")
