Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: VPS26 knockout HAP1 cell lysate (20 µg)
Lane 3: Jurkat cell lysate (20 µg)
Lanes 1 - 3: Merged signal (red and green). Green - ab23892 observed at 40 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab23892 was shown to recognize VPS26 when VPS26 knockout samples were used, along with additional cross-reactive bands. Wild-type and VPS26 knockout samples were subjected to SDS-PAGE. ab23892 at a concentration of 1 µg/ml and ab8245 (loading control to GAPDH) diluted to 1/10000 were incubated overnight at 4°C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-VPS26 antibody (ab23892) at 1 µg/ml

Lane 1 : Human kidney lysate
Lane 2 : Human kidney lysate with Human VPS26 peptide (ab24288) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Alexa Fluor Goat polyclonal to Rabbit IgG (700) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 40 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?
Additional bands at: 25 kDa, 50 kDa. We are unsure as to the identity of these extra bands.

ab23892 detects a band of ~ 38kDa in Human kidney lysate. This band is quenched by the addition of the immunizing peptide, ab24288. ab23892 also detects a band of the correct size in HEK293 and mouse kidney lysate (data not shown).

ICC/IF image of ab23892 stained human HEK 293 cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab23892, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa and HepG2 cells.

All lanes : Anti-VPS26 antibody (ab23892) at 1/1000 dilution

Lane 1 : Rat NRK whole cell lysate at 25 µg
Lane 2 : Rat NRK whole cell lysate at 50 µg

Secondary
All lanes : HRP-conjugated goat anti-rabbit polyclonal IgG at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 40 kDa
Observed band size: 40 kDa


Exposure time: 10 seconds


Blocked with 5% milk for 30 min at 25°C

See Abreview

Immunofluorescence analysis of HeLa cells treated with mDpy-30 siRNA, staining VPS26 (green) with ab23892.

Cells were fixed formaldehyde, permeabilized and blocked with 5% goat serum for 20 minutes. Samples were incubated with primary antibody in blocking buffer for 2 hours before incubating with fluorophore-conjugated secondary antibody for 1 hour. Nuclei were detected using DAPI.

All lanes : Anti-VPS26 antibody (ab23892) at 1/1000 dilution

Lane 1 : HEK293T whole cell lysate
Lane 2 : HeLa whole cell lysate

Lysates/proteins at 50 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit polyclonal IgG at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 40 kDa
Observed band size: 40 kDa


Exposure time: 2 seconds


Blocked with 5% milk for 30 min at 25°C

See Abreview

Immunocytochemical analysis of Rat NRK cell line, labeling VPS26 with ab23892. Cells were formaldehyde fixed, permeabilized with 0.1% Triton X-100 in PBS for 3 minutes and blocked with 1% BSA for 30 minutes at 25°C. Cells were incubated with ab23892 diluted 1/250 in 1% BSA in PBS for 1 hour at 25°C.

See Abreview

Anti-VPS26 antibody (ab23892) at 1/1000 dilution + COS whole cell lysate, African Green Monkey at 50 µg with Milk, 30 minutes, 25°C at 5 %

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 40 kDa


Exposure time: 10 seconds

See Abreview