All lanes : Anti-EFEMP1/Fibulin-3 antibody [EPR22855-4] (ab256457) at 1/1000 dilution

Lane 1 : HUVEC (human umbilical vein endothelial cell) whole cell lysate
Lane 2 : Human eye tissue lysate
Lane 3 : Human ovary lysate
Lane 4 : Human skeletal muscle lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 54 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

The molecular weight observed is consistent with what has been described in the literature (PMID: 28740094).

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

Exposure Time: Lane 1:103 seconds; Lane 2:8 seconds; Lane 3:15 seconds; Lane 4:48 seconds.

EFEMP1/Fibulin-3 was immunoprecipitated from 0.35 mg HUVEC (human umbilical vein endothelial cell) whole cell lysate with ab256457 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256457 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: HUVEC (human umbilical vein endothelial cell) whole cell lysate 10ug

Lane 2: ab256457 IP in HUVEC whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab256457 in HUVEC whole cell lysate

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

Exposure time: 1 second

 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HUVEC (human umbilical vein endothelial cell) cells labelling EFEMP1/Fibulin-3 with ab256457 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730)  isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HUVEC (human umbilical vein endothelial cell) cells labelling EFEMP1/Fibulin-3 with ab256457 at 1/500 dilution, followed by Ab256457 anti-EFEMP1 Fibulin-3 ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HUVEC cell line is observed. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling EFEMP1/Fibulin-3 with ab256457 at 1/500 dilution (1.12 ug/ml) followed by 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. Positive staining on the basement membrane in human testes. The section was incubated with ab256457 for 15 mins at RT. 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).

 

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling EFEMP1/Fibulin-3 with ab256457 at 1/500 dilution (1.12 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the stromal cells of human colon.  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. The section was incubated with ab256457 for 15 mins at RT. 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).

 

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling EFEMP1/Fibulin-3 with ab256457 at 1/500 dilution (1.12 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the stromal cells of human colon carcinoma. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. The section was incubated with ab256457 for 15 mins at RT. 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).

 

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling EFEMP1/Fibulin-3 with ab256457 at 1/500 dilution (1.12 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the stroma cells of human breast carcinoma (PMID/ 19115204). The section was incubated with ab256457 for 15 mins at RT. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. 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).