Anti-SND1 antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-SND1 antibody (AB65078)

ICC/IF image of ab65078 stained Hek293 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab65078, 1µg/ml) overnight at +4°C. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa, HepG2, and MCF-7 cells at 1µg/ml, and in 100% Methanol fixed (5 min) HeLa, Hek293, HepG2, and MCF-7 cells at 1µg/ml.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SND1 antibody (AB65078)

IHC image of ab65078 staining SND1 in Human normal breast formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab65078, 5µg/ml, for 15 mins 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.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IP

Unknown

Immunoprecipitation - Anti-SND1 antibody (AB65078)

SND1 was immunoprecipitated using 0.5mg Ramos whole cell extract, 5µg of Rabbit polyclonal to SND1 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, Ramos whole cell extract 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 ab65078.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 102kDa : SND1.

All lanes:

Immunoprecipitation - Anti-SND1 antibody (ab65078)

Predicted band size: 102 kDa

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• WB

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Western blot - Anti-SND1 antibody (AB65078)

All lanes:

Western blot - Anti-SND1 antibody (ab65078) at 1 µg/mL

Lane 1:

Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

HeLa Whole Cell Lysate - Hydroxyurea Treated (48hr, 2µM) at 10 µg

Lane 3:

HeLa Whole Cell Lysate - Staurosporine Treated (24hr, 500nM) at 10 µg

Lane 4:

TE 671 (Human Rhabdomyosarcoma) Whole Cell Lysate at 10 µg

Lane 5:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 6:

JEG-3 (Human placental choriocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 7:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 102 kDa

Observed band size: 102 kDa

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