Ubiquitin Monobody IP Beads & Buffer Kit

Cosmo Bio

Catalog No.:
CSR-MB-001-SAB
Ubiquitin Monobody IP Beads & Buffer Kit
Figure 1. Immunoprecipitation Using Mammalian Cells Lysate was prepared from untreated human HCT116 cells according to the protocol described in this datasheet. Immunoprecipitation was performed using monobody-immobilized beads with 50 µL of lysate. Ubiquitinated proteins bound to the beads were eluted with 50 µL of 1× LDS Elution Buffer. To evaluate the recovery efficiency of ubiquitinated proteins, the eluted fraction was diluted to match the volume of the lysate and flow-through fractions, and each sample was subjected to SDS-PAGE using a 4–12% Bis-Tris gel. The separated proteins were transferred onto a PVDF membrane and blocked with 3% skim milk. Ubiquitinated proteins were detected using a mouse monoclonal anti-ubiquitin primary antibody (P4D1, 1:1000 dilution) and an HRP-conjugated goat polyclonal anti-mouse IgG secondary antibody (1:2000 dilution), followed by ECL-based chemiluminescence detection. Lane 1: Molecular weight marker Lane 2: Lysate Lane 3: Flow-through Lane 4: Eluate
Figure 2. Comparison of Signal Patterns by Detection Method Using eluted fractions obtained from the same immunoprecipitation assay as described in Figure 1, three PVDF blots were prepared, each employing a different combination of blocking reagent, primary antibody, and secondary reagent (see lane descriptions below). Ubiquitinated proteins were detected on each blot using the ECL method. Notably, in lanes 3 and 4, in addition to the ubiquitinated proteins, the Anti-Pan Ubiquitin Monobody, Biotin dissociated from the streptavidin beads was also detected (arrowhead), highlighting differences in signal patterns depending on the detection system. Lane 1: Molecular weight marker Lane 2: 3% skim milk / anti-ubiquitin mouse monoclonal antibody (P4D1, 1:1000 dilution) / HRP conjugated goat anti-mouse IgG polyclonal antibody (1:2000 dilution) Lane 3: 3% BSA / Anti-Pan Ubiquitin Monobody, Biotin (1:5000 dilution) / HRP-conjugated anti-6xHis mouse monoclonal antibody (1:2000 dilution) Lane 4: 3% BSA / Anti-Pan Ubiquitin Monobody, Biot
Figure 3. Broad Applicability Across Species (A) Lysates were prepared using the lysis buffer included in this kit from human HCT116 cells (Lane 2), Arabidopsis leaves (Lane 3), and Rhizopus hyphae (Lane 4). For Arabidopsis and Rhizopus, lysates were pretreated by gel filtration prior to protein quantification. Each lysate was adjusted to 1 mg/mL, and 5 μg of protein was separated by SDS-PAGE followed by CBB staining. This allowed visual confirmation that the protein concentrations were approximately equivalent across the samples. Lane 1: Molecular weight marker (B) Immunoprecipitation, SDS-PAGE, and western blotting were performed following the same protocol as in Figure 1, except that no dilution was applied to the eluted fractions. Lane 1: Molecular weight marker Lanes 2–4: Lysate, flow-through, and eluate from human HCT116 cells Lanes 5–7: Lysate, flow-through, and eluate from Arabidopsis leaves Lanes 8–10: Lysate, flow-through, and eluate from Rhizopus hyphae
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Product Description

This product is a dedicated support kit designed to simplify and enhance the efficiency of immunoprecipitation (IP) using Anti-Pan Ubiquitin Monobody, Biotin (Cat. No. MB-001-B). It includes streptavidin magnetic beads, along with a set of buffers optimized for each step of the workflow, including lysate preparation, monobody immobilization, capture of ubiquitinated proteins, washing, and elution.
 
The Anti-Pan Ubiquitin Monobody, Biotin binds to ubiquitinated proteins regardless of ubiquitin chain length or linkage type, making it well suited for a range of downstream applications, including Western blot and proteomic analyses. Furthermore, because the amino acid sequence of ubiquitin is highly conserved across species, immunoprecipitation using Anti-Pan Ubiquitin Monobody, Biotin is applicable to a wide range of organisms.
 
A notable feature of this kit is its use of FG beads®—streptavidin magnetic beads coated with poly(glycidyl methacrylate). As a result, nonspecific adsorption, which is often problematic with conventional beads, is minimized, enabling the high-purity, low-background recovery of ubiquitinated proteins.



Documents & Links for Ubiquitin Monobody IP Beads & Buffer Kit
Datasheet Ubiquitin Monobody IP Beads & Buffer Kit Datasheet

Documents & Links for Ubiquitin Monobody IP Beads & Buffer Kit
Datasheet Ubiquitin Monobody IP Beads & Buffer Kit Datasheet
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