Immunohistochemical staining of paraffin embedded human gliocytoma with purified ab32136 at a working dilution of 1/500. The secondary antibody used is ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

All lanes : Anti-Amyloid Precursor Protein antibody [Y188] (ab32136) at 1/20000 dilution (purified)

Lane 1 : HeLa cell lysate
Lane 2 : Human fetal brain tissue lysate
Lane 3 : HEK293 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 87 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

ab32136 staining Amyloid Precursor Protein in wild-type HEK293 cells (top panel) and APP knockout HEK293 cells (ab255362) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 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 with ab32136 at 1/500 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Immunofluorescence staining of SH-SY5Y cells with purified ab32136 at a working dilution of 1 in 100, counter-stained with DAPI. Tubulin was stained with mouse anti-tubulin at a dilution of 1/1000 (ab7291) and Alexa Fluor^® 594 goat anti-mouse at a dilution of 1/500 (ab150120) . The secondary antibody was ab150077 Alexa Fluor^® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in the bottom middle and right hand panels - for the first negative control, purified ab32136 was used at a dilution of 1/200 followed by an Alexa Fluor^® 555 goat anti-mouse antibody at a dilution of 1/500 and for the second negative control mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab15007) were used.

ab32136 (purified) at 1/30 immunoprecipitating amyloid beta precursor protein in A431 (Lane 1). Lane 2 - PBS. For western blotting a HRP-conjugated anti-rabbit IgG specific to the non-reduced form of IgG was used as the secondary antibody (1/1500). Blocking buffer and concentration: 5% NFDM/TBST. Diluting buffer and concentration: 5% NFDM /TBST.

Unpurified ab32136, at a 1/250 dilution, staining Amyloid beta precursor protein by immunohistochemistry.
Positive immunohistochemical staining, using paraffin embedded human brain tissue (A).
Negative immunohistochemical staining, using human breast (B), skeletal muscle (C) and liver (D) tissues.
Tissues were stained in parallel on the same Normal Tissue Array.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

All lanes : Anti-Amyloid Precursor Protein antibody [Y188] (ab32136) at 1/20000 dilution (purified)

Lane 1 : Mouse brain tissue lysate
Lane 2 : Rat brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 87 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Anti-Amyloid Precursor Protein antibody [Y188] (ab32136) at 1/20000 dilution (unpurified) + Hela cell lysate

Predicted band size: 87 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?
Additional bands at: 110 kDa. We are unsure as to the identity of these extra bands.

Anti-Amyloid Precursor Protein antibody [Y188] (ab32136) at 1/1000 dilution (unpurifed) + Mouse spleen whole cell lysate at 100 µg

Secondary
HRP-conjugated goat anti-rabbit polyclonal IgG at 1/4000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 87 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?
Additional bands at: 125 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 10 minutes


Blocked with 5% milk

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