Anti-PRPF8/Prp8 antibody

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PRPF8/Prp8 antibody (AB79237)

IHC image of PRPF8/Prp8 staining in human colon carcinoma 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 ab79237, 10μg/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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PRPF8/Prp8 antibody (AB79237)

ICC/IF image of ab79237 stained HeLa cells. The cells were 100% methanol fixed (5 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 (ab79237, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.

• IP

Unknown

Immunoprecipitation - Anti-PRPF8/Prp8 antibody (AB79237)

PRPF8/Prp8 was immunoprecipitated using 0.5mg Hela whole cell extract, 5μg of Rabbit polyclonal to PRPF8/Prp8 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, Hela 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 ab79237.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 250kDa; PRPF8/Prp8

All lanes:

Immunoprecipitation - Anti-PRPF8/Prp8 antibody (ab79237)

Predicted band size: 274 kDa

false

• WB

Unknown

Western blot - Anti-PRPF8/Prp8 antibody (AB79237)

The 250 kDa band observed is comparable to the molecular weight seen with other commercially available antibodies to PRPF8/Prp8.

All lanes:

Western blot - Anti-PRPF8/Prp8 antibody (ab79237) at 1 µg/mL

Lane 1:

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

Lane 2:

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

Lane 3:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Lane 4:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 5:

MEF1 (Mouse embryonic fibroblast 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: 274 kDa

Observed band size: 250 kDa

true