Anti-SESN2/Sestrin-2 antibody [EPR18907] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SESN2/Sestrin-2 antibody [EPR18907] - BSA and Azide free (AB236025)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (Human colorectal carcinoma cell line) cells labeling SESN2/Sestrin-2 with ab178518 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HCT116 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/250 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178518).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SESN2/Sestrin-2 antibody [EPR18907] - BSA and Azide free (AB236025)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HCT 116 (Human colorectal carcinoma cell line) cells labeling SESN2/Sestrin-2 with ab178518 at 1/60 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178518).

• IP

Supplier Data

Immunoprecipitation - Anti-SESN2/Sestrin-2 antibody [EPR18907] - BSA and Azide free (AB236025)

SESN2/Sestrin-2 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab178518 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab178518 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate, 10μg (Input).

Lane 2 : ab178518 IP in HeLa whole cell lysate.

Lane 3 : Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab178518 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

SESN2/Sestrin-2 expression is low in HeLa cells and can be enriched through immunoprecipitation.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178518).

All lanes:

Immunoprecipitation - Anti-SESN2/Sestrin-2 antibody [EPR18907] (<a href='/en-us/products/primary-antibodies/sesn2-sestrin-2-antibody-epr18907-ab178518'>ab178518</a>)

Predicted band size: 54 kDa

false

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SESN2/Sestrin-2 antibody [EPR18907] - BSA and Azide free (AB236025)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling SESN2/Sestrin-2 with ab178518 at 1/60 dilution (red) compared with a 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 (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178518).

• WB

Lab

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] - BSA and Azide free (AB236025)

This data was developed using the same antibody clone in a different buffer formulation (ab178518).

Lanes 1-4 : Merged signal (red and green). Green - ab178518 observed at 54 kDa. Red - loading control ab8245 observed at 36 kDa.

ab178518 Anti-SESN2/Sestrin-2 antibody [EPR18907] was shown to specifically react with 2 in wild-type HeLa cells. Loss of signal was observed when knockout sample ab257665 was used. Wild-type and 2 knockout samples were subjected to SDS-PAGE. ab178518 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours 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 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (<a href='/en-us/products/primary-antibodies/sesn2-sestrin-2-antibody-epr18907-ab178518'>ab178518</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

SESN2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SESN2 (Sestrin-2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-sesn2-sestrin-2-knockout-hela-cell-line-ab265669'>ab265669</a>)

Lane 3:

LoVo cell lysate at 20 µg

Lane 4:

HEK-293T cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 38 kDa,54 kDa

Observed band size: 48 kDa,54 kDa

false

• WB

Lab

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] - BSA and Azide free (AB236025)

This data was developed using the same antibody clone in a different buffer formulation (ab178518).

Lanes 1-3 : Merged signal (red and green). Green - ab178518 observed at 54 kDa. Red - loading control ab8245 observed at 36 kDa.

ab178518 Anti-SESN2/Sestrin-2 antibody [EPR18907] was shown to specifically react with SESN2/Sestrin-2 in wild-type HeLa cells. Loss of signal was observed when knockout sample ab257665 was used. Wild-type and SESN2/Sestrin-2 knockout samples were subjected to SDS-PAGE. ab178518 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 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (<a href='/en-us/products/primary-antibodies/sesn2-sestrin-2-antibody-epr18907-ab178518'>ab178518</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

SESN2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SESN2 (Sestrin-2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-sesn2-sestrin-2-knockout-hela-cell-line-ab265669'>ab265669</a>)

Lane 3:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 54 kDa

Observed band size: 54 kDa

false

• WB

Lab

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] - BSA and Azide free (AB236025)

False colour image of Western blot : Anti-SESN2/Sestrin-2 antibody [EPR18907] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab178518 was shown to bind specifically to SESN2/Sestrin-2. A band was observed at 54 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in SESN2 knockout cell line ab269486 (knockout cell lysate ab269650). To generate this image, wild-type and SESN2 knockout HEK-293 cell lysates were analysed. First, 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 IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (<a href='/en-us/products/primary-antibodies/sesn2-sestrin-2-antibody-epr18907-ab178518'>ab178518</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 cell lysate at 20 µg

Lane 2:

SESN2 knockout HEK-293 cell lysate at 20 µg

Lane 2:

Western blot - Human SESN2 (Sestrin-2) knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-sesn2-sestrin-2-knockout-hek-293-cell-line-ab269486'>ab269486</a>)

Predicted band size: 54 kDa

Observed band size: 54 kDa

false