Mouse / IgG2b
Anti-SUMO1 antibody, ASM11, is a SUMO1 mouse monoclonal antibody that is part of the Signal-Seeker™ product line. Anti-SUMO1 mouse monoclonal antibody was raised against full-length recombinant SUMO1 protein (Uniprot: P63165). The antibody has been shown to recognize a broad profile of SUMO1 proteins in HAP1, HeLa, and 293 cell lysates (Fig. 1A) and to detect sub-nanogram amounts of recombinant SUMO1 (Fig. 1B). The antibody shows high specificity (Fig. 1A WT vs KO lane).with no cross reactivity to SUMO2 (Fig. 1B). Furthermore, the sensitivity of ASM01 was compared to commercially available SUMO1 antibodies, and the data shows that ASM01 detects a more robust and specific profile of SUMO1 modified proteins (Fig. 1C). ASM01 is purified by Protein G affinity chromatography and is supplied as a lyophilized white powder.
Each Lot of antibody is quality controlled to provide a high batch to batch consistency. The Lot specific µg per tube can be found in the Lot specific COA documents.
Figure 1: Western Blot using SUMO-2/3 Antibody
ASM01 was used at a 1:5000 dilution (0.2 µg/mL) following the recommended western blot protocol (see above). Figure 1A: HAP1 wildtype (WT) or SUMO1 knockout (KO) lysate, HeLa cell +/- NEM lysate and 293 cell +/- NEM lysate was obtained using BlastR lysis and filter system. 10 µg of each lysate were separated by SDS-PAGE and transferred to PVDF. Robust SUMO1 profiles were detected for every cell type. Specificity is shown by the lack of SUMO1 detection in SUMO1 KO cells and significantly diminished profiles in lysates prepared in the absence of the SENP inhibitor (NEM).
Figure 1B: Titration of recombinant SUMO1 lanes 1-6 contain 5.0, 2.5, 1.25, 0.6, 0.3, and 0.15 ng SUMO1, while lane 7 contains 1000 ng of recombinant SUMO2.
Figure 1C: HAP1 WT and KO lysate as prepared in 1A was used. 10 µg of each lysate were separated by SDS-PAGE and transferred to PVDF. SUMO1 proteins were detected using the recommended concentrations of ASM01 (Cytoskeleton), 21C7 (Invitrogen—purified), 21C7 (DSHB—supernatant), and D11 (Santa Cruz) as shown in the figure. All samples were developed and imaged simultaneously to ensure identical experimental conditions. To see the full Western blot protocol, see the product datasheet.
Figure 2: Immunoprecipitation using SUMO-2/3 Antibody
HAP1 wildtype (WT) and knockout (KO) cells were prepared in BlastR buffer and filter system supplemented with NEM and PIC02. Figure 2A: 1 mg of lysate per reaction was used to immunoprecipitate (IP) SUMO1 modified proteins using 40 µg of 5D8 antibody (ASM01), 40 µg of ASM11-beads, 40 µg of 21C7 (DSHB-supernatant), or 40 µg of mouse IgG control antibody. 5D8, 21C7 supernatant, and mouse IgG were bound to Protein G Agarose as described in the method (see above). Enriched SUMO1 samples were analyzed by western blot using ASM01 antibody at 1:5000, and mouse Trubelot Ultra-HRP secondary at 1:1000 in 5% milk. Trueblot secondary was used to minimize heavy and light chain detection.
Figure 2B: IP was performed using ASM11 as in Fig 2A. SUMO1 modified proteins were visualized with ASM01 1:5000, and anti-mouse secondary at 1:20,000 to highlight the profile of SUMOylated proteins in the region between 64-30 kDa that may be masked by heavy and light chain interference when using unconjugated antibodies for IP. To see the full Immunoprecipitation protocol, see the product datasheet.
Immunofluorescence applications for ASM01 have not been tested
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Signal-Seeker™ SUMOylation 1 Detection Kit (Cat. # BK165)
Signal-Seeker™ SUMOylation 1 Affinity Beads (Cat.# ASM11-beads)
Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)
|Kim, Catherine et al.||SUMOylation of mitofusins: A potential mechanism for perinuclear mitochondrial congression in cells treated with mitochondrial stressors||Biochimica et Biophysica Acta - Molecular Basis of Disease||2021||ISSN 1879-260X|