Immunohistochemical analysis of paraffin-embedded human pancreatic carcinoma tissue (A) and the paired adjacent pancreas tissue (B) labeling Glypican 1/ GPC1 with ab255600 at 1/500 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic and membranous staining on human pancreatic carcinoma (panel A) and no staining on the paired adjacent pancreas (panel B, PMID: 28440066) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is 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.

The section was incubated with ab255600 for 30 mins at RT.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue (A) and the paired adjacent lung tissue (B) labeling Glypican 1/ GPC1 with ab255600 at 1/500 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human lung carcinoma (panel A) and the paired adjacent lung (panel B) was barely stained (PMID: 29785020) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is 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.

The section was incubated with ab255600 for 30 mins at RT.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemical analysis of paraffin-embedded esophagus lung carcinoma tissue labeling Glypican 1/ GPC1 with ab255600 at 1/500 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on esophagus carcinoma (PMID: 28445969) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is 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.

The section was incubated with ab255600 for 30 mins at RT.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Glypican 1/ GPC1 was immunoprecipitated from 0.35 mg DU 145 (human prostate carcinoma cell line) whole cell lysate 10μg with ab255600 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab255600 1/1000 dilution (0.65 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane  1: DU 145 (human prostate carcinoma cell line) whole cell lysate 10μg

Lane 2: ab255600 IP in DU 145 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255600 in DU 145 whole cell lysate.

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

Exposure time: 75 seconds.

All lanes : Anti-Glypican 1/ GPC1 antibody [EPR22580-72] (ab255600) at 1/1000 dilution

Lane 1 : DU 145 (human prostate carcinoma cell line) cultured under serum-free conditions, whole cell lysate
Lane 2 : PC-3 (human prostate adenocarcinoma epithelial cell line) whole cell lysate
Lane 3 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate
Lane 4 : MDA-MB-231 (human breast adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 62 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Blocking and dilution biffer: 5% NFDM/TBST.

Molecular weight observed is consistent with what has been described in the literature (PMID: 8207484, 27313791).

Homology with other Glypican family members (38.88% to GPC-2; 21.55% to GPC-3; 43.41% to GPC-4; 25.86% to GPC-5; 45.05% to GPC-6).

Exposure time:

Lanes 1-2: 5.5 seconds

Lanes 3-4: 3 minutes