SUMO-2/3 Antibody Mouse Monoclonal (Clone 12F3)

SUMO-2/3 Antibody Mouse Monoclonal (Clone 12F3)
$0.00

Host/Isotype
Mouse / IgG2a-kappa

Clone
12F3

Species Reactivity
Broad Reactivity

Validation Data
White Paper
Antibodypedia

Anti-SUMO2/3 antibody is a SUMO-2/3 mouse monoclonal antibody that is part of the Signal-Seeker™ product line. Anti-SUMO-2/3 mouse monoclonal antibody was raised against full-length recombinant SUMO-2 protein (Uniprot: P61956) combined with a proprietary mix of peptides that include CQIRFRFDGQPINE. The antibody has been shown to recognize a wide range of SUMO-2/3-targeted proteins in HeLa cell lysate (Fig. 1B) and to detect sub-nanogram amounts of recombinant SUMO-2 (Fig. 1A). Epitope mapping has identified that the antibody recognizes a sequence/structure within the peptide CQIRFRFDGQPINE. The peptide sequence is conserved in mammals, birds, and amphibians, giving the antibody broad species reactivity. ASM23 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

12F3 was used for immunoblotting (1:500 dilution) following the recommended Western blot protocol (see below). Figure 1A: Titration of recombinant SUMO-2 (40-0.6 ng) and SUMO-1 (800 ng). SUMO-2 was detected down to 0.6 ng while SUMO-1 was not detected at 800 ng. Figure 1B: Induction of SUMOylation by heat shock and reduction of SUMOylation by SUMO-2 shRNA knockdown. Cell lysates were prepared from HeLa cells: Lane 2: Heat Shock treated (43°C  for 10min), Lane 3: untreated, Lane 4:  shRNA SUMO-2 knock down. 20 µg of HeLa cell lysates were used for each sample. Lane 1: position of molecular weight markers. Figure 1C: Specificity of SUMO-2 knockdown signal. Lane 1: parental HeLa cell lysates, Lane 2: SUMO-2 shRNA control lysates, Lane 3: SUMO-1 shRNA knock-down cell lysates, Lane 4: SUMO-2 shRNA knock-down cell lysates. Arrow head indicates free SUMO-2/3. To see the full Western blot protocol, see the product datasheet.

Figure 2: Immunoprecipitation using SUMO-2/3 Antibody

Denatured cell lysates were prepared from HS43, CT37 and KD S212. 1mg of lysate was used for the immunoprecipitation of SUMO-2/3 conjugates. IP experiments were performed by the protocol presented in IP Method. Western blots of immunoprecipitated proteins were developed using 12F3 (A) or anti-TFII-I antibody (B). (A) Star (*) and circle (o) indicate heavy and light chains of antibodies. Unconjugated free SUMO is denoted by triangle. (B) Unconjugated TFII-I is visible near 120 kDa. Multiple bands indicate that TFII-I is SUMOylated by several SUMO-2/3 proteins.  TFII-I has previously been reported to be a target for Sumoylation .  To see the full Immunoprecipitation protocol, see the product datasheet.

Figure 3: Immunofluorescence of HeLa cells in metaphase with SUMO-2/3 Antibody

HeLa cells were stained and visualized by confocal fluorescence microscopy as described in the IF method below. The cells were stained against α/β-tubulin (sheep anti-tubulin Ab, Cat# ATN02, green) and SUMO-2/3 (12F3, red). DNA was stained with DAPI. Mitotic cells in metaphase were imaged with a Zeiss LSM 780 confocal microscope (1.4 NA 63X objective).  Detection of SUMO 2/3 at chromosomes can be observed during mitosis as has been previously reported10. To see the full Immunofluorescence protocol, see the product datasheet.

Figure 4: Immunofluorescence of HeLa cells in interphase with SUMO-2/3 Antibody

HeLa cells were stained and visualized by widefield fluorescence microscopy as described in the IF method below. The cells were stained against α/β-tubulin (sheep anti-tubulin Ab, Cat# ATN02, green) and SUMO-2/3 (12F3, red). DNA was stained with DAPI. Cells in interphase were imaged with a Zeiss Axio Observer.Z1 microscope (1.4 NA 63X objective). PML nuclear bodies (nuclear dots) were visible in SUMO-2/3 staining as has been previously reported11.  To see the full Immunofluorescence protocol, see theproduct datasheet.

For more information contact:  signalseeker@cytoskeleton.com

Associated Products:

Signal-Seeker™ SUMOylation 2/3 Detection Kit (Cat. # BK162)

Signal-Seeker™ SUMOylation 2/3 Affinity Beads (Cat.# ASM24-beads)

Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)

For product Datasheets and MSDSs please click on the PDF links below.

Sample Size Datasheet (Cat. ASM23-S):  

Certificate of Analysis:  Lot 011

 

For the most recent publications citing this and other Signal-Seeker™ products, see our Signal-Seeker™ Validation Data Page click here

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If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com