All lanes : Anti-CD39 antibody [EPR22507-48] (ab227840) at 1/1000 dilution

Lane 1 : Mouse placenta lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse spleen lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 58 kDa
Observed band size: 78 kDa why is the actual band size different from the predicted?


Exposure time: 81 seconds

The molecular weight observed is consistent with what has been described in the literature (PMID:18036594).

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling CD39 with ab227840 at 1/500 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on mouse spleen (PMID: 18567823). The section was incubated with ab227840 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse splenocytes labeling CD39 with ab227840 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in subsets of mouse splenocyte. The nuclear stain is DAPI (blue). Counterstained with Anti-CD45R/B220 Antibody (Alexa Fluor^® 647)  at a 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling CD39 with ab227840 at 1/500 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on blood vessels of mouse liver (PMID: 18567823). The section was incubated with ab227840 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Flow cytometric analysis of mouse splenocytes labeling CD39 with ab227840 at 1/500. Cells were stained with rabbit IgG Isotype control (ab172730) (Left panel) or ab227840 (Right panel). Then stained with anti-CD45R conjugated to Alexa Fluor^® 647. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

CD39 was immunoprecipitated from 0.35 mg mouse placenta lysate with ab227840 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab227840 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: Mouse placenta lysate 10 µg (Input).
Lane 2: ab227840 IP in mouse placenta lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab227840 in mouse placenta lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 1 second.