All lanes : Anti-Annexin-4/ANXA4 antibody [EPR22929-208] (ab256456) at 1/2000 dilution

Lane 1 : C6 (rat glial tumor glial cell) whole cell lysate
Lane 2 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 4 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 35 kDa
Observed band size: 31,36 kDa why is the actual band size different from the predicted?

The expression molecular weight observed is consistent with what has been described in the literature (PMID: 16893879). Blocking/Diluting buffer and concentration: 5% NFDM/TBST.  Exposure Time: 15 seconds.

All lanes : Anti-Annexin-4/ANXA4 antibody [EPR22929-208] (ab256456) at 1/2000 dilution

Lane 1 : Human kidney lysate
Lane 2 : Human liver lysate
Lane 3 : Human lung lysate
Lane 4 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 5 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 6 : Mouse liver lysate
Lane 7 : Mouse kidney lysate
Lane 8 : Mouse lung lysate
Lane 9 : Rat kidney lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-3 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 4-9 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 35 kDa
Observed band size: 31,36 kDa why is the actual band size different from the predicted?

The expression molecular weight observed is consistent with what has been described in the literature (PMID: 16893879). Blocking/Diluting buffer and concentration: 5% NFDM/TBST.  Exposure Time: Lanes 1-4: 59 seconds; Lanes 5-8: 125 seconds; Lane 9: 10 seconds.

Immunohistochemical analysis of paraffin-embedded rat stomach tissue labeling Annexin-4/ANXA4 with ab256456 at 1/5000 dilution (0.103 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in rat stomach. The section was incubated with ab256456 for 10 mins at RT. The immunostaining staining 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 citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse stomach tissue labeling Annexin-4/ANXA4 with ab256456 at 1/5000 dilution (0.103 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in mouse stomach. The section was incubated with ab256456 for 10 mins at RT. The immunostaining staining 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 citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labeling Annexin-4/ANXA4 with ab256456 at 1/5000 dilution (0.103 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human endometrium (PMID: 16954445) is observed. The section was incubated with ab256456 for 10 mins at RT. The immunostaining staining 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 citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Annexin-4/ANXA4 with ab256456 at 1/5000 dilution (0.103 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human kidney (PMID: 9720986) is observed. The section was incubated with ab256456 for 10 mins at RT. The immunostaining staining 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 citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human pancreas tissue labeling Annexin-4/ANXA4 with ab256456 at 1/5000 dilution (0.103 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in centroacinar cells and islets of human pancreas. The section was incubated with ab256456 for 10 mins at RT. The immunostaining staining 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 citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Annexin-4/ANXA4 with ab256456 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled 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.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling Annexin-4/ANXA4 with ab256456 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled 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.

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized rat pancreas tissue labeling Annexin-4/ANXA4 with ab256456 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Positive staining on mouse pancreas (PMID: 25176043) is observed. The nuclear counter stain is 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.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse pancreas tissue labeling Annexin-4/ANXA4 with ab256456 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Positive staining on mouse pancreas (PMID: 25176043) is observed. The nuclear counter stain is 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.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized rat kidney tissue labeling Annexin-4/ANXA4 with ab256456 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Positive staining on rat kidney (PMID: 15145526) is observed. The nuclear counter stain is 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.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse kidney tissue labeling Annexin-4/ANXA4 with ab256456 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Positive staining on mouse kidney (PMID: 15145526) is observed. The nuclear counter stain is 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.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling Annexin-4/ANXA4 with ab256456 at 1/100 5.2 µg/ml dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing nuclear, cytoplasmic and staining in NIH/3T3 cell line. 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 2 µg/ml dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labeling Annexin-4/ANXA4 with ab256456 at 1/100 5.2 µg/ml dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing nuclear, cytoplasmic and staining in HepG2 cell line. 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 2 µg/ml dilution.