Anti-FHL2 antibody [EPR17860-23]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-FHL2 antibody [EPR17860-23] (AB202586)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A-673 (Human muscle Ewing's Sarcoma cell line) cells labeling FHL2 with ab202586 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic and weakly nuclear staining on A-673 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/500 dilution (red).

The negative controls are as follows :
-ve control 1 : ab202586 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/500 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-FHL2 antibody [EPR17860-23] (AB202586)

ab202586 staining FHL2 in wild-type U2OS cells (top panel) and FHL2 knockout U2OS cells (bottom panel). The cells were fixed with 4% PFA (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab202586 at 1/100 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-FHL2 antibody [EPR17860-23] (AB202586)

ab202586 staining FHL2 in wild-type U2OS cells (top panel) and FHL2 knockout U2OS cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab202586 at 1/100 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IP

Supplier Data

Immunoprecipitation - Anti-FHL2 antibody [EPR17860-23] (AB202586)

FHL2 was immunoprecipitated from 1mg of SW480 (Human colorectal adenocarcinoma cell line) whole cell lysate with ab202586 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab202586 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : SW480 whole cell lysate 10 μg (Input). Lane 2 : ab202586 IP in SW480 whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab202586 in SW480 whole cell lysate.

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

Exposure time : 5 seconds.

All lanes:

Immunoprecipitation - Anti-FHL2 antibody [EPR17860-23] (ab202586)

Predicted band size: 32 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-FHL2 antibody [EPR17860-23] (AB202586)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embyro fibroblast cells) cells labeling FHL2 with ab202586 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic and weakly nuclear staining on A-673 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/500 dilution (red).

The negative controls are as follows :
-ve control 1 : ab202586 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/500 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution.

• WB

Supplier Data

Western blot - Anti-FHL2 antibody [EPR17860-23] (AB202586)

5% NFDM/TBST : Blocking and diluting buffer.

All lanes:

Western blot - Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/2000 dilution

Lane 1:

SW480 (Human colorectal adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

Lane 3:

HT1080 (Human fibrosarcoma cells) whole cell lysate at 10 µg

Lane 4:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/1000 dilution

Predicted band size: 32 kDa

Observed band size: 32 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-FHL2 antibody [EPR17860-23] (AB202586)

** Lanes 1-3 : ** Merged signal (red and green). Green - ab202586 observed at 32 kDa. Red - loading control ab7291 observed at 50 kDa. ab202586 Recombinant Anti-FHL2 antibody [EPR17860-23] was shown to specifically react with FHL2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265475 (knockout cell lysate ab257441) was used. Wild-type and FHL2 knockout samples were subjected to SDS-PAGE. ab202586 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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-FHL2 antibody [EPR17860-23] (ab202586) at 1/1000 dilution

Lane 1:

Wild-type HeLa lysate at 20 µg

Lane 2:

FHL2 knockout HeLa lysate at 20 µg

Lane 2:

Western blot - Human FHL2 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-fhl2-knockout-hela-cell-line-ab265475'>ab265475</a>)

Lane 3:

K562 lysate at 20 µg

Predicted band size: 32 kDa

Observed band size: 32 kDa

false

• WB

Lab

Western blot - Anti-FHL2 antibody [EPR17860-23] (AB202586)

Lanes 1 - 3 : Merged signal (red and green). Green - ab202586 observed at 32 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab202586 was shown to react with FHL2 in wild-type U-2 OS cells in western blot with loss of signal observed in FHL2 knockout sample. Wild-type and FHL2 knockout U-2 OS cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab202586 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/1000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 40 µg

Lane 2:

FHL2 knockout U-2 OS cell lysate at 40 µg

Lane 2:

Western blot - Human FHL2 knockout U-2 OS cell line (<a href='/en-us/products/cell-lines/human-fhl2-knockout-u-2-os-cell-line-ab262496'>ab262496</a>)

Lane 3:

HeLa cell lysate at 40 µg

Predicted band size: 32 kDa

Observed band size: 32 kDa

false

• WB

Supplier Data

Western blot - Anti-FHL2 antibody [EPR17860-23] (AB202586)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/10000 dilution

All lanes:

Human fetal heart lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 32 kDa

Observed band size: 32 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-FHL2 antibody [EPR17860-23] (AB202586)

Lanes 1 - 3 : Merged signal (red and green). Green - ab202586 observed at 32 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab202586 was shown to react with FHL2 in wild-type U-2 OS cells in western blot with loss of signal observed in FHL2 knockout sample. Wild-type and FHL2 knockout U-2 OS cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab202586 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/1000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 40 µg

Lane 2:

FHL2 knockout U-2 OS cell lysate at 40 µg

Lane 3:

HeLa cell lysate at 40 µg

Predicted band size: 32 kDa

false

• WB

Supplier Data

Western blot - Anti-FHL2 antibody [EPR17860-23] (AB202586)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/10000 dilution

Lane 1:

Mouse heart lysate at 10 µg

Lane 2:

Mouse kidney lysate at 10 µg

Lane 3:

Rat heart lysate at 10 µg

Lane 4:

Rat kidney lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/1000 dilution

Predicted band size: 32 kDa

Observed band size: 32 kDa

false

Exposure time: 30s