All lanes : Anti-Protein S antibody [EPR24509-70] (ab280885) at 1/1000 dilution

Lane 1 : Huh7 (human hepatocellar carcinoma epithelial cell) whole cell lysate
Lane 2 : Human liver tissue lysate
Lane 3 : Human lung tissue lysate
Lane 4 : Human stomach tissue lysate
Lane 5 : HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate
Lane 6 : HaCaT (human skin keratinocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 75 kDa
Observed band size: 85,75,65 kDa why is the actual band size different from the predicted?

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

The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID:28118606, 27447084).

Negative control: HaCaT (PMID:28118606).

The blot of lane 1 was developed using a higher sensitivity ECL substrate.

Exposure time: Lane 1: 92 seconds Lane 2: 48 seconds Lane 3: 26 seconds Lane 4-6: 3 minutes

 

Immunohistochemical analysis of paraffin-embedded human testis tissue labelling Protein S with ab280885 at 1/1000 dilution (1.603 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the leydig cells in human testis (PMID: 7945211). The section was incubated with ab280885 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: 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 solution2) for 20 mins.

Protein S was immunoprecipitated from 0.35 mg of Human liver tissue lysate with ab280885 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280885 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Human liver tissue lysate 10 ug

Lane 2: ab280885 IP in Human liver tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab280885 in human liver tissue lysate

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

Exposure time: 32 seconds

 

Anti-Protein S antibody [EPR24509-70] (ab280885) at 1/2000 dilution + Human plasma at 20 µg

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

Predicted band size: 75 kDa
Observed band size: 85,75 kDa why is the actual band size different from the predicted?

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

The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID:28118606, 27447084).

Exposure time: 15 seconds

 

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling Protein S with ab280885 at 1/1000 dilution (1.603 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the plasma in human colon. The section was incubated with ab280885 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: 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 solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labelling Protein S with ab280885 at 1/1000 dilution (1.603 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: no staining on human pancreas (PMID:7822769). The section was incubated with ab280885 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: 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 solution2) for 20 mins.