All lanes : Anti-Robo1 antibody [EPR23699-159] (ab256791) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : ROBO1 knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 181 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab256791).

Lanes 1 - 4: Merged signal (red and green). Green - ab256791 observed at 120 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab256791 was shown to react with Robo1 in western blot. The band observed in the knockout lysate lane below 120 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab256791 and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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 h at room temperature before imaging.

This data was developed using ab256791, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of LoVo (Human colorectal adenocarcinoma epithelial cell) (Left) / HeLa (Human cervix adenocarcinoma epithelial cell) (Right) cells labelling Robo1 with ab256791 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: LoVo(PMID: 16740745).

Gated on viable cells.

All lanes : Anti-Robo1 antibody [EPR23699-159] (ab256791) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : LoVo (human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 4 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 181 kDa
Observed band size: 120/130 kDa why is the actual band size different from the predicted?

This data was developed using ab256791, the same antibody clone in a different buffer formulation.

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 20471383, 16740745). The 120/130-kDa bands correspond to the Robo1 cleavage fragments from metalloprotease and/or gamma secretase activities (PMID: 20570941, 20471383).

Negative control: LoVo (PMID: 16740745); Low expression: MCF7 (PMID: 16740745).

Lysates used in this blot have experienced a freeze-thaw cycle.

Exposure time: 10 seconds.

This data was developed using ab256791, the same antibody clone in a different buffer formulation.

Robo1 was immunoprecipitated from 0.35 mg Mouse E14.5 brain tissue lysate with ab256791 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256791 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/1000 dilution.

Lane 1: Mouse E14.5 brain tissue lysate 10 ug.

Lane 2: ab256791 IP in Mouse E14.5 brain tissue lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab256791 in mouse E14.5 brain tissue lysate.

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

Exposure time: 1 second.

 

All lanes : Anti-Robo1 antibody [EPR23699-159] (ab256791) at 1/1000 dilution

Lane 1 : Mouse E14.5 brain tissue lysate
Lane 2 : C57 E18 brain tissue lysate
Lane 3 : Rat E14 brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 181 kDa
Observed band size: 120/250 kDa why is the actual band size different from the predicted?

This data was developed using ab256791, the same antibody clone in a different buffer formulation.

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

The 120 kDa band corresponds to the Robo1 cleavage fragments from metalloprotease and/or gamma secretase activities (PMID: 20570941, 20471383).

Lysates used in this blot have experienced a freeze-thaw cycle.

Exposure time: 70 seconds.

All lanes : Anti-Robo1 antibody [EPR23699-159] (ab256791) at 1/1000 dilution

Lane 1 : His-tagged human Robo1 recommbinant protein (aa660-897(Q883R))
Lane 2 : His-tagged human Robo2 recommbinant protein (aa621-859)
Lane 3 : His-tagged human Robo3 recommbinant protein (aa655-892)
Lane 4 : His-tagged human Robo4 recommbinant protein (aa301-463)

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 181 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

This data was developed using ab256791, the same antibody clone in a different buffer formulation.

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

These recombinant proteins were made in house. The sample loaded onto lane 1 was purified Robo1 recombinant protein expressed from a mammalian - HEK-293 expression system. The samples loaded onto lanes 2-4 were E.coil extracts containing Robo2, Robo3 or Robo4 recombinant protein respectively.

Exposure time: 26 seconds.