All lanes : Anti-Asialoglycoprotein Receptor 1/HL-1 antibody [EPR22642-17] (ab254262) at 1/1000 dilution

Lane 1 : Human adult liver tissue lysate
Lane 2 : Human brain tissue lysate
Lane 3 : HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-2 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lane 3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 33 kDa
Observed band size: 40-50 kDa why is the actual band size different from the predicted?

Negative control: human brain.

The diffuse bands between 40-50 kDa are likely due to glycosylation (PMID:30965276).

The expression profile is consistent with what has been described in the literature (PMID:27241665, 29858079).

Blocking/diluting buffer and concentration: 5% NFDM/TBST

Asialoglycoprotein Receptor 1/HL-1 antibody was immunoprecipitated from 0.35 mg HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate 10μg with ab254262 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254262 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate 10μg

Lane 2: ab254262 IP in HepG2 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254262 in HepG2 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 7 seconds.

The band at around 100kDa is described as dimers (PMID: 7663158).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell line) cells labelling Asialoglycoprotein Receptor 1/HL-1 antibody with ab254262 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous and weak cytoplasmic staining in HepG2 cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)  was used to counter stain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Asialoglycoprotein Receptor 1/HL-1 antibody with ab254262 at 1/1000 dilution (0.5 μg/ml)  followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human liver (PMID:23979840, 26422581, 27241665) is observed.

The section was incubated with ab254262 for 15 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. 

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

Immunohistochemical analysis of paraffin-embedded Human liver cancer tissue labeling Asialoglycoprotein Receptor 1/HL-1 antibody with ab254262 at 1/1000 dilution (0.5 μg/ml)  followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human liver cancer (PMID:23979840,26422581,27241665) is observed.

The section was incubated with ab254262 for 15 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

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).

Negative control: No staining in human colon (PMID:23979840).

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Asialoglycoprotein Receptor 1/HL-1 antibody with ab254262 at 1/1000 dilution (0.5 μg/ml)  followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

The section was incubated with ab254262 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.  Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

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).

Negative control: No staining in human cerebrum (PMID:23979840)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Asialoglycoprotein Receptor 1/HL-1 antibody with ab254262 at 1/1000 dilution (0.5 μg/ml)  followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

The section was incubated with ab254262 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.  Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

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).

Flow cytometric analysis of HepG2 (Human hepatocellular carcinoma epithelial cell line) cells labelling Asialoglycoprotein Receptor 1/HL-1 antibody with ab254262 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.