Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (AB52636)

Immunocytochemical analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized PC-12 (Rat adrenal gland pheochromocytoma) cells labeling Synaptophysin with ab52636 at 1/250 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining in PC-12 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (AB52636)

Overlay histogram showing PC12 cells stained with ab52636 (red line). The cells were fixed with methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52636, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (0.5μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in PC12 cells fixed with 4% paraformaldehyde (10 min) used under the same conditions.Please note that Abcam do not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.

• WB

Lab

Western blot - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (AB52636)

Lanes 1-3 : Merged signal (red and green). Green - ab52636 observed at 38 kDa. Red - loading control, ab7291 observed at 50 kDa.

ab52636 Anti-Synaptophysin antibody [EP1098Y] was shown to specifically react with Synaptophysin in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267272 (knockout cell lysate ab257060) was used. Wild-type and Synaptophysin knockout samples were subjected to SDS-PAGE. ab52636 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (ab52636) at 1/1000 dilution

Lane 1:

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

Lane 2:

SYP knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human SYP (Synaptophysin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-syp-synaptophysin-knockout-hek-293t-cell-line-ab267272'>ab267272</a>)

Lane 3:

Human brain tissue lysate

Predicted band size: 33 kDa

Observed band size: 38 kDa

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• WB

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Western blot - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (AB52636)

Lanes 1-3 : Merged signal (red and green). Green - ab52636 observed at 38 kDa. Red - loading control, ab7291 observed at 50 kDa.

ab52636 Anti-Synaptophysin antibody [EP1098Y] was shown to specifically react with Synaptophysin in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267272 (knockout cell lysate ab257060) was used. Wild-type and Synaptophysin knockout samples were subjected to SDS-PAGE. ab52636 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (ab52636) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

Syp knockout HEK293T cell lysate at 20 µg

Lane 3:

Human brain tissue lysate at 20 µg

Predicted band size: 33 kDa

Observed band size: 38 kDa

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• WB

Lab

Western blot - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (AB52636)

Blocking/Diluting buffer and concentration : 5% NFDM /TBST

All lanes:

Western blot - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (ab52636) at 0.203 µg/mL

Lane 1:

Human brain tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Rat brain lysate tissue lysate at 20 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 0.05 µg

Predicted band size: 33 kDa

Observed band size: 38 kDa

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• WB

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Western blot - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (AB52636)

All lanes:

Western blot - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (ab52636) at 1/200000 dilution

All lanes:

Rat brain membrane

Secondary

All lanes:

Goat anti-rabbit HRP labeled at 1/2000 dilution

Predicted band size: 33 kDa

Observed band size: 38 kDa

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• WB

CiteAb

Western blot - Anti-Synaptophysin antibody [EP1098Y] - Synaptic Marker (AB52636)

Synaptophysin western blot using anti-Synaptophysin antibody [EP1098Y] ab52636. Publication image and figure legend from Zhou, J., Liu, Z., et al., 2015, Int J Neuropsychopharmacol, PubMed 26364272.

ab52636 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab52636 please see the product overview.

Western-blot analysis of the 4 fractions (Hom, Syn, Ves, and synaptic junction) obtained by biochemical fractionation. The fractions were analyzed using antibodies against synaptophysin (synaptic vesicle marker), Stx1a (synaptic active zone marker), and PSD95 (postsynaptic density marker).

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