Anti-MTAP antibody [EPR22570-76] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTAP antibody [EPR22570-76] - BSA and Azide free (AB256356)

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in human breast (PMID : 26751376, 18712977) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254265).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MTAP antibody [EPR22570-76] - BSA and Azide free (AB256356)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (human lung carcinoma cell line) and HT-29 (human colorectal adenocarcinoma cell line) cells labeling MTAP with ab254265 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and nuclear staining in HT-29 cell line. The nuclear counter stain is DAPI (blue) Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

SEcondary antibody only control : Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Negative control : A549 PMID : 8971171.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254265).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTAP antibody [EPR22570-76] - BSA and Azide free (AB256356)

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in stroma cells and no staining in tumor cells of human bladder cancer (PMID : 27270441) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254265).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MTAP antibody [EPR22570-76] - BSA and Azide free (AB256356)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized A549 (human lung carcinoma cell line) (Left) anf H-29 (human colorectal adenocarcinoma cell line) (Right) cells labeling MTAP with ab254265 at 1/400 dilution (red) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

Negative control : A549 (PMID : 8971171).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254265).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTAP antibody [EPR22570-76] - BSA and Azide free (AB256356)

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in rat liver (PMID : 15492751) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254265).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTAP antibody [EPR22570-76] - BSA and Azide free (AB256356)

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in mouse liver (PMID : 15492751) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254265).

• WB

Lab

Western blot - Anti-MTAP antibody [EPR22570-76] - BSA and Azide free (AB256356)

This data was developed using the same antibody clone in a different buffer formulation (ab254265).

Lanes 1-4 : Merged signal (red and green). Green - ab254265 observed at 32 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab254265 Anti-MTAP antibody [EPR22570-76] was shown to specifically react with MTAP in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265272 (knockout cell lysate ab257194) was used. Wild-type and MTAP knockout samples were subjected to SDS-PAGE. ab254265 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MTAP antibody [EPR22570-76] (<a href='/en-us/products/primary-antibodies/mtap-antibody-epr22570-76-ab254265'>ab254265</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MTAP knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human MTAP knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-mtap-knockout-hela-cell-line-ab265272'>ab265272</a>)

Lane 3:

HT-29 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 31 kDa

Observed band size: 32 kDa

false

• WB

Lab

Western blot - Anti-MTAP antibody [EPR22570-76] - BSA and Azide free (AB256356)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254265).

Western blot : Anti-MTAP antibody [EPR22570-76] ab254265 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 31kDa in Wild-type HeLa cell lysates with no signal observed at this size in MTAP knockout HeLa cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-MTAP antibody [EPR22570-76] (<a href='/en-us/products/primary-antibodies/mtap-antibody-epr22570-76-ab254265'>ab254265</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysates at 20 µg

Lane 2:

MTAP knockout HeLa cell lysates at 20 µg

Lane 3:

HepG2 cell lysates at 20 µg

Lane 4:

A549 cell lysates at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 31 kDa

Observed band size: 31 kDa

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