Anti-U2AF65抗体

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-U2AF65 antibody (AB37530)

IHC image of ab37530 staining in Human breast adenocarcinoma 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 ab37530, 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-U2AF65 antibody (AB37530)

ab37530 staining U2AF65 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab37530 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 ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). 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

Lab

Immunoprecipitation - Anti-U2AF65 antibody (AB37530)

U2AF65 was immunoprecipitated using 0.5mg SHSY5Y whole cell extract, 5µg of Rabbit polyclonal to U2AF65 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, SHSY5Y 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 ab37530.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 65kDa : U2AF65.

All lanes:

Immunoprecipitation - Anti-U2AF65 antibody (ab37530)

Predicted band size: 54 kDa

false

• WB

Ap

Western blot - Anti-U2AF65 antibody (AB37530)

Although the predicted band size is 53kDa based on Swiss-prot data, a band of 65kDa has been previously observed. J Biol Chem. 2004 Nov 26;279(48) : 49773-9 (PMID : 15377657)

All lanes:

Western blot - Anti-U2AF65 antibody (ab37530) at 1/250 dilution

Lane 1:

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

Lane 2:

Western blot - Jurkat whole cell lysate (<a href='/products/cell-lysates/jurkat-whole-cell-lysate-ab7899'>ab7899</a>) at 10 µg

Lane 3:

Western blot - A-431 whole cell lysate (<a href='/products/cell-lysates/a-431-whole-cell-lysate-ab7909'>ab7909</a>) at 10 µg

Lane 4:

Western blot - HEK-293 whole cell lysate (<a href='/products/cell-lysates/hek-293-whole-cell-lysate-ab7902'>ab7902</a>) at 10 µg

Lane 5:

Hep G2 whole cell lysate (<a href='/products/unavailable/hep-g2-whole-cell-lysate-ab7900'>ab7900</a>) at 10 µg

Lane 6:

MCF-7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 7:

SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate at 10 µg

Lane 8:

U2OS (Human osteosarcoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Rabbit IgG secondary antibody (ab28446) at 1/10000 dilution

Predicted band size: 54 kDa

Observed band size: 65 kDa

false

• WB

Unknown

Western blot - Anti-U2AF65 antibody (AB37530)

All lanes:

Western blot - Anti-U2AF65 antibody (ab37530) at 1/250 dilution

Lane 1:

Marker

Lane 2:

Zebrafish brain homogenate at 20 µg

Lane 3:

Zebrafish heart homogenate at 10 µg

Lane 4:

Zebrafish liver homogenate at 10 µg

Lane 5:

Zebrafish skeletal muscle homogenate at 10 µg

Lane 6:

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

Secondary

All lanes:

Goat polyclonal to Rabbit IgG – H&L – Pre-Adsorbed (HRP) at 1/6000 dilution

Predicted band size: 54 kDa

Observed band size: 53 kDa

true

Exposure time: 5min