Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: UCHL1 (PGP9.5) knockout HAP1 whole cell lysate (20 µg)
Lane 3: Hek293 whole cell lysate (20 µg)
Lane 4: Ms brain whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab27053 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab27053 was shown to recognize UCHL1 (PGP9.5) in wild type cells as signal was lost at the expected MW in UCHL1 (PGP9.5) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and UCHL1 (PGP9.5) knockout samples were subjected to SDS-PAGE. Ab27053 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 dilution respectively. Blots were developed 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 1/10000 dilution for 1 hour at room temperature before imaging.

IHC image of Anti-PGP9.5 antibody staining in a section of formalin-fixed paraffin-embedded normal human pancreas performed on a Leica BOND™ system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab27053, 1 µg/mL, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

All lanes : Anti-PGP9.5 antibody (ab27053) at 1 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)
Lane 2 : Brain (Mouse) Tissue Lysate (ab27253)
Lane 3 : Brain (Rat) Tissue Lysate (ab7942)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 25 kDa
Observed band size: 25 kDa
Additional bands at: 37 kDa (possible non-specific binding), 60 kDa (possible non-specific binding), 75 kDa (possible non-specific binding)


Exposure time: 10 seconds

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with abX overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

 

Abcam recommends using milk as the blocking agent.

ICC/IF image of ab27053 stained PC12 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab27053, 5µg/ml) overnight at +4°C. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

All lanes : Anti-PGP9.5 antibody (ab27053) at 1 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 3 : Brain (Mouse) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 25 kDa
Observed band size: 25 kDa
Additional bands at: 65 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 1 minute

PGP9.5 was immunoprecipitated using 0.5mg Mouse Brain tissue lysate, 5µg of Rabbit polyclonal to PGP9.5 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Brain tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab27053.
Secondary: Clean-Blot IP Detection Reagent (HRP) at 1/500 dilution.
Band: 26kDa, non specific band - 65kDa: We are unsure as to the identity of this extra band; PGP9.5

Anti-PGP9.5 antibody (ab27053) at 1/250 dilution + Recombinant Human PGP9.5 protein (ab82628) at 0.01 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 25 kDa


Exposure time: 3 minutes