Anti-Amyloid Precursor Protein antibody [Y188] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Amyloid Precursor Protein antibody [Y188] - BSA and Azide free (AB256586)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32136).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Amyloid Precursor Protein antibody [Y188] - BSA and Azide free (AB256586)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32136).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Amyloid Precursor Protein antibody [Y188] - BSA and Azide free (AB256586)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32136).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Amyloid Precursor Protein antibody [Y188] - BSA and Azide free (AB256586)

This data was developed using the same antibody clone in a different buffer formulation (ab32136).

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).

• IP

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Immunoprecipitation - Anti-Amyloid Precursor Protein antibody [Y188] - BSA and Azide free (AB256586)

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.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32136).

All lanes:

Immunoprecipitation - Anti-Amyloid Precursor Protein antibody [Y188] (<a href='/en-us/products/primary-antibodies/amyloid-precursor-protein-antibody-y188-ab32136'>ab32136</a>)

Predicted band size: 86 kDa

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• WB

Lab

Western blot - Anti-Amyloid Precursor Protein antibody [Y188] - BSA and Azide free (AB256586)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32136). Western blot : Anti-APP antibody [Y188] (ab32136) staining at 1/20000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32136 was shown to bind specifically to APP. A band was observed at 80-130 kDa in wild-type HAP1 and HEK-293T cell lysates with no signal observed at this size in both APP knockout cell lines. To generate this image, wild-type and APP knockout HAP1 and HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Lanes 1 - 7:

Western blot - Anti-Amyloid Precursor Protein antibody [Y188] (<a href='/en-us/products/primary-antibodies/amyloid-precursor-protein-antibody-y188-ab32136'>ab32136</a>) at 1/20000 dilution

Lanes 1 - 7:

Western blot - Anti-Amyloid Precursor Protein antibody [Y188] - BSA and Azide free (ab256586)

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

APP knockout HAP1 cell lysate at 20 µg

Lane 3:

Wild-type HEK-293T cell lysate at 20 µg

Lane 4:

APP knockout HEK-293T cell lysate at 20 µg

Lane 5:

Human Hippocampus cell lysate at 20 µg

Lane 6:

U-87 MG cell lysate at 20 µg

Lane 7:

K562 cell lysate at 20 µg

Observed band size: 80-130 kDa

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