Anti-PGP9.5 antibody - Neuronal Marker

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PGP9.5 antibody - Neuronal Marker (AB27053)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PGP9.5 antibody - Neuronal Marker (AB27053)

ab27053 staining PGP9.5 in PC12 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes 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 ab27053 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PGP9.5 antibody - Neuronal Marker (AB27053)

ab27053 staining PGP9.5 in primary rat neurons/glia, DIV14 (prepared from E18 rat hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. SDHEP) cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes 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 ab27053 at 5µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• IP

Unknown

Immunoprecipitation - Anti-PGP9.5 antibody - Neuronal Marker (AB27053)

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

All lanes:

Immunoprecipitation - Anti-PGP9.5 antibody - Neuronal Marker (ab27053)

Predicted band size: 24 kDa

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• WB

Unknown

Western blot - Anti-PGP9.5 antibody - Neuronal Marker (AB27053)

All lanes:

Western blot - Anti-PGP9.5 antibody - Neuronal Marker (ab27053) at 1 µg/mL

Lane 1:

Human brain tissue lysate - total protein (<a href='/en-us/products/unavailable/human-brain-tissue-lysate-total-protein-ab29466'>ab29466</a>) at 10 µg

Lane 2:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Lane 3:

Brain (Mouse) Tissue Lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 24 kDa

Observed band size: 25 kDa,65 kDa

true

Exposure time: 1min

• WB

Lab

Western blot - Anti-PGP9.5 antibody - Neuronal Marker (AB27053)

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.

All lanes:

Western blot - Anti-PGP9.5 antibody - Neuronal Marker (ab27053)

Predicted band size: 24 kDa

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• WB

Lab

Western blot - Anti-PGP9.5 antibody - Neuronal Marker (AB27053)

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 ab27053 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.

All lanes:

Western blot - Anti-PGP9.5 antibody - Neuronal Marker (ab27053) at 1 µg/mL

Lane 1:

Human brain tissue lysate - total protein (<a href='/en-us/products/unavailable/human-brain-tissue-lysate-total-protein-ab29466'>ab29466</a>) at 10 µg

Lane 2:

Brain (Mouse) Tissue Lysate (ab27253) at 10 µg

Lane 3:

Brain (Rat) Tissue Lysate (<a href='/en-us/products/unavailable/brain-rat-tissue-lysate-ab7942'>ab7942</a>) at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 24 kDa

Observed band size: 25 kDa,37 kDa,60 kDa,75 kDa

true

Exposure time: 10s

• WB

Unknown

Western blot - Anti-PGP9.5 antibody - Neuronal Marker (AB27053)

All lanes:

Western blot - Anti-PGP9.5 antibody - Neuronal Marker (ab27053) at 1/250 dilution

All lanes:

Recombinant Human PGP9.5 protein (<a href='/en-us/products/unavailable/recombinant-human-pgp95-protein-ab82628'>ab82628</a>) at 0.01 µg

Secondary

All lanes:

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

Predicted band size: 24 kDa

true

Exposure time: 3min