All lanes : Anti-FHL1 antibody [EPR22842-95] (ab255828) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : FHL1 knockout HeLa cell lysate
Lane 3 : HEK293T cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 36 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab255828 observed at 32 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab255828 Anti-FHL1 antibody [EPR22842-95] was shown to specifically react with FHL1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab266011 (knockout cell lysate ab257952) was used. Wild-type and FHL1 knockout samples were subjected to SDS-PAGE. ab255828 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.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling FHL1 with ab255828 at 1/500 dilution (1.036 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the stroma and smooth muscle in human colon is observed. The section was incubated with ab255828 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NCI-H1299 (Human lung carcinoma epithelial cell) cells labeling FHL1 with ab255828 at 1/50 10 µg/ml dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing cytoplasmic and nuclear staining in NCI-H1299 cell line. Low expression control: NCI-H460 cell line (PMID: 21702045). ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 2 µg/ml dilution.

All lanes : Anti-FHL1 antibody [EPR22842-95] (ab255828) at 1/1000 dilution

Lane 1 : NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysate
Lane 2 : NCI-H460 (human large cell lung cancer epithelial cell) whole cell lysate
Lane 3 : Mouse skeletal muscle lysate
Lane 4 : Human skeletal muscle lysate
Lane 5 : Human lung lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lanes 4-5 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 36 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

There are five isoforms of FHL1. The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 21702045).

Low expression control: NCI-H460 (PMID: 21702045).

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

Exposure Time:

Lanes 1-2: 3 minutes;

Lane 3: 26 seconds;

Lane 4: 1 second;

Lane 5: 26 seconds.

All lanes : Anti-FHL1 antibody [EPR22842-95] (ab255828) at 1/1000 dilution

Lane 1 : Mouse kidney lysate
Lane 2 : Mouse spleen lysate
Lane 3 : Rat heart lysate
Lane 4 : Rat skeletal muscle lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 36 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

There are five isoforms of FHL1. The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 21702045).

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

Exposure Time:

Lanes 1-3: 3 minutes;

Lane 4: 3 seconds.

FHL1 was immunoprecipitated from 0.35 mg mouse skeletal muscle lysate 10µg with ab255828 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab255828 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse skeletal muscle lysate 10µg.

Lane 2: ab255828 IP in mouse skeletal muscle lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255828 in mouse skeletal muscle lysate.

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

Exposure time/ 30 seconds.

 

FHL1 was immunoprecipitated from 0.35 mg human lung lysate 10µg with ab255828 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab255828 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Human lung lysate 10µg.

Lane 2: ab255828 IP in human lung lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255828 in human lung lysate.

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

Exposure time/ 30 seconds.

 

Immunohistochemical analysis of paraffin-embedded rat skeletal muscle tissue labeling FHL1 with ab255828 at 1/500 dilution (1.036 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on rat skeletal muscle (PMID: 27443559, 9714789) is observed. The section was incubated with ab255828 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling FHL1 with ab255828 at 1/500 dilution (1.036 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on mouse lung (PMID: 29067108) is observed. The section was incubated with ab255828 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human cardiac muscle tissue labeling FHL1 with ab255828 at 1/500 dilution (1.036 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human cardiac muscle (PMID: 9714789) is observed. The section was incubated with ab255828 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling FHL1 with ab255828 at 1/500 dilution (1.036 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human skeletal muscle (PMID: 27443559, 9714789) is observed. The section was incubated with ab255828 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.