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?

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.

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.

Anti-Robo1 antibody [EPR23699-159] (ab256791) at 1/1000 dilution + HeLa whole cell lysate at 20 µg

Secondary
VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 181 kDa
Observed band size: 130,250 kDa why is the actual band size different from the predicted?


Exposure time: 92 seconds

Buffer and concentration: 5% NFDM/TBST

250 kDa full length band and 130 kDa cleavage fragment band were ovserved.
Freshly prepared lysate was used in this blot.

Robo1 was immunoprecipitated from 0.35 mg HepG2 whole cell lysate with ab256791 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256791 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1:  HepG2 whole cell lysate 10 ug.

Lane 2: ab256791 IP in HepG2 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab256791 in HepG2 whole cell lysate.

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

Exposure time: 93 seconds.

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?

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.

 

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?

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?

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.