ASM23

antibody from Cytoskeleton, Inc.

Targeting: SUMO2 SMT3B, SMT3H2

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

ASM23

Provider

Cytoskeleton, Inc.

Product name

Anti-SUMO-2/3

Antibody type

Monoclonal

Antigen

Full-length recombinant SUMO-2 protein (Uniprot: P61956) combined with a proprietary mix of peptides that include CQIRFRFDGQPINE.

Description

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 at www.cytoskeleton.com.

Host

Mouse

Conjugate

Unconjugated

Epitope

CQIRFRFDGQPINE

Isotype

IgG

Antibody clone number

12F3

Vial size

2 x 100 µl

Concentration

Specified in COA

Storage

Lyophilized 4 degrees C. Reconstituted -20 degrees C.

Handling

Shipped at room temperture. The lyophilized protein can be stored desiccated at 4 degrees C for 6 months. Store at -20 degrees C when reconstituted.

Western blot

Supportive validation

Submitted by

Cytoskeleton, Inc. (provider)

Main image

Experimental details

12F3 was used for immuno-blotting (1:500 dilution) following the recommended Western blot protocol (see below). Figure 1A: Titrations 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. 20µg 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.

Sample type

Cell lysates were prepared from HeLa cells

Primary Ab dilution

1:500

Secondary Ab

goat anti mouse secondary antibody

Secondary Ab dilution

1:20,000

Protocol

Protocol

Immunocytochemistry

Supportive validation

Submitted by

Cytoskeleton, Inc. (provider)

Main image

Experimental details

HeLa cells were stained and visualized by confocal fluorescence microscopy as described in the IF method below. The cells were stained against ?/?-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). Enrichment 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.

Sample type

HeLa cells

Primary Ab dilution

1:500

Secondary Ab

fluorescently-labeled donky anti-mouse

Secondary Ab dilution

1:500

Protocol

Protocol

Supportive validation

Submitted by

Cytoskeleton, Inc. (provider)

Main image

Experimental details

HeLa cells were stained and visualized by widefield fluorescence microscopy as described in the IF method below. The cells were stained against ?/?-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 the product datasheet.

Sample type

HeLa cells

Primary Ab dilution

1:500

Secondary Ab

fluorescently-labeled donky anti-mouse

Secondary Ab dilution

1:500

Protocol

Protocol

Immunoprecipitation

Supportive validation

Submitted by

Cytoskeleton, Inc. (provider)

Main image

Experimental details

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 120kDa. 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.

Sample type

Denatured cell lysates were prepared from HS43, CT37 and KD S2

Primary Ab dilution

Assay Dependent

Secondary Ab

Secondary Ab

Protocol

Protocol