ab45338 staining LYRIC in HeLa cells. The cells were fixed with 100% methanol (5min), 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 ab45338 at 10μg/ml and ab7291, Anti-alpha Tubulin antibody [DM1A] - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed (shown in green) and ab1500120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

 

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

All lanes : Anti-LYRIC/AEG1 antibody (ab45338) at 1 µg/ml

Lane 1 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 2 : MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 3 : MDA-MB-361 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 4 : DU145 (Human Prostate carcinoma epithelial like cell line) Whole Cell lysate
Lane 5 : PC-3 whole cell lysate (ab3954)
Lane 6 : U-87 MG nuclear extract lysate (ab14903)

Lysates/proteins at 10 µg per lane.

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

Performed under reducing conditions.

Predicted band size: 64 kDa
Observed band size: 75 kDa

why is the actual band size different from the predicted?
Additional bands at: 130 kDa, 80 kDa (possible post-translational modification). We are unsure as to the identity of these extra bands.

All lanes : Anti-LYRIC/AEG1 antibody (ab45338) at 1 µg/ml

Lane 1 : Skeletal Muscle (Mouse) Tissue Lysate
Lane 2 : Heart (Mouse) Tissue Lysate
Lane 3 : Skeletal Muscle (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 64 kDa


Exposure time: 1 minute

IHC image of LYRIC/AEG1 staining in Human skin cancer formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab45338, 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 image of ab45338 stained MCF7 cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab45338, 5µg/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). This antibody also gave a positive IF result in MCF7 cells fixed in 100% methanol (10 min) cells.