Anti-Syntaxin 1a antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Syntaxin 1a antibody (AB41453)

ICC/IF image of ab41453 stained rat PC12 cells. The cells were methanol fixed (5 min), permabilised in PBS-T (20 min) and incubated with the antibody (ab41453, 1μg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Syntaxin 1a antibody (AB41453)

ab41453 staining Syntaxin 1a in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. 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 ab41453 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 ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IP

Unknown

Immunoprecipitation - Anti-Syntaxin 1a antibody (AB41453)

Syntaxin 1a was immunoprecipitated using 0.5mg Mouse Brain tissue lysate, 5µg of Rabbit polyclonal to Syntaxin 1a 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 ab41453.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 33kDa, non specific band - 25kDa : We are unsure as to the identity of this extra band; Syntaxin 1a

All lanes:

Immunoprecipitation - Anti-Syntaxin 1a antibody (ab41453)

Predicted band size: 33 kDa

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• WB

Project2941****

Western blot - Anti-Syntaxin 1a antibody (AB41453)

All lanes:

Western blot - Anti-Syntaxin 1a antibody (ab41453) 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) Mouse Tissue Lysate at 10 µg

Lane 3:

Brain (Rat) Rat Tissue Lysate at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

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