ab24733 staining GC1q R in HeLa 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 ab24733 at 5µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed 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).

All lanes : Anti-GC1q R antibody [60.11] (ab24733) at 5 µg/ml

Lane 1 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 4 : Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate
Lane 5 : Human liver tissue lysate - total protein (ab29889)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?
Additional bands at: 68 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 3 minutes

Lane 1 : Mw standards
Lanes 2-3 : Anti-GC1q R antibody [60.11] (ab24733) at 1/2000 dilution

Lane 2 : 293T whole cell lysate at 20 µg
Lane 3 : Hela whole cell lysate at 20 µg

Secondary
Lanes 2-3 : Alexa Fluor® 680 conjugated goat anti-mouse

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds


The blot was blocked with 5% milk for 1 hour at 25°C prior to incubating with the primary antibody for 13 hours at 4°C.

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ab24733 used in Flow Cytometry.
U937 cells were cultured in RPMI 10% FCS, treated with PMA (5nM) and, 6-12 hours later, cells were harvested, spinned and resuspended to 200,000 cells in 100µl. ab24733 used at 10µg/ml for 30 minutes at 4°C. A phycoerythrin conjugated goat anti-mouse polyclonal was used as the secondary antibody at a 1/100 dilution.

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Overlay histogram showing HeLa cells stained with ab24733 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab24733, 0.5μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150117) at 1/4000 dilution for 30 min at 22°C.

Isotype control antibody (black line) was mouse IgG1 [15-6E10A7] (ab170190, 0.5μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

IHC image of ab24733 staining in human tonsil 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 ab24733, 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.

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.