Recombinant Anti-alpha Actinin/ACTN1 antibody [EP2527Y] (ab68194)

Purified ab68194 at 1:20 dilution (2µg) immunoprecipitating alpha Actinin/ACTN1 in NIH/3T3 whole cell lysate.
Lane 1 (input): NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10µg
Lane 2 (+): ab68194 + NIH/3T3 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab68194 in NIH/3T3 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP)(ab131366) (1:5000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 103 kDa

Blocking buffer: 5% NFDM/TBST

Immunofluorescence staining of Actinin/ACTN1 using ab68194 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 3 (left panel), 5 (middle panel) and 10 days (right panel) post induction.

The cells were fixed with 100% MeOH (5 min) 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 overnight at +4°C with ab68194 at 0.5 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

Blocking buffer: 5% NFDM/TBST

Lanes 1- 2: Merged signal (red and green). Green - ab68194 observed at 103 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab68194 was shown to react with alpha Actinin/ACTN1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265610 (knockout cell lysate ab257337) was used. Wild-type HeLa and ACTN1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab68194 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 2000 dilution and a 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.

Immunofluorescence staining of Actinin/ACTN1 using ab68194 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 3 (left panel), 5 (middle panel) and 10 days (right panel) post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins 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 overnight at +4°C with ab68194 at 0.5 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Alpha Actinin/ACTN1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: NIH 3T3 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab68194 observed at 103 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab68194 was shown to specifically react with alpha Actinin/ACTN1 in wild-type HAP1 cells as signal was lost in alpha Actinin/ACTN1 knockout cells. Wild-type and alpha Actinin/ACTN1 knockout samples were subjected to SDS-PAGE. ab68194 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.