All lanes : Anti-Prion protein PrP antibody [EP1802Y] (ab52604) at 1/5000 dilution (Purified)

Lane 1 : Human brain lysate
Lane 2 : Mouse brain lysate
Lane 3 : Rat brain lysate
Lane 4 : SK-MEL-28 (Human malignant melanoma ) whole cell lysate
Lane 5 : Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 28 kDa
Observed band size: 20-37 kDa why is the actual band size different from the predicted?

The molecular weights observed represent different glycosation states and are consistent with what has been described in the literature (PMID: 20670940, PMID: 19568430, PMID: 15240877 and PMID: 22558368).

Blocking/Diluting buffer: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebrum tissue sections labeling Prion protein PrP with purified ab52604 at 1/500 dilution (1.32 µg/mL). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 1 (pH 6.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Flow Cytometry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells, labeling Prion protein PrP with Purified ab52604 at 1:70 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Anti-Prion protein PrP antibody [EP1802Y] (ab52604) at 1/5000 dilution (unpurified) + Recombinant Mouse Prion protein PrP (ab74056) at 0.01 µg

Secondary
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: 28 kDa


Exposure time: 10 seconds

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab52604 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebrum tissue sections labeling Prion protein PrP with purified ab52604 at 1/500 dilution (1.32 µg/mL). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 1 (pH 6.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Overlay histogram showing SH-SY5Y cells stained with unpurified ab52604 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52604, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebrum tissue sections labeling Prion protein PrP with purified ab52604 at 1/500 dilution (1.32 µg/mL). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 1 (pH 6.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemical analysis of brain glioma using ab52604 (unpurified) at a dilution of 1/100. Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.