Recombinant Anti-POMC antibody [EPR22534-165] (ab254257)

Lanes 1-4 : Anti-POMC antibody [EPR22534-165] (ab254257) at 1/5000 dilution
Lanes 5-6 : Anti-POMC antibody [EPR22534-165] (ab254257) at 1/1000 dilution

Lane 1 : Mouse pituitary lysate
Lane 2 : Mouse liver lysate
Lane 3 : Human pituitary lysate
Lane 4 : Human liver lysate
Lane 5 : Rat pituitary lysate
Lane 6 : Rat liver 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: 29 kDa
Observed band size: 35, 16 kDa why is the actual band size different from the predicted?

Exposure time:
Lanes 1-2: 3 seconds;
Lanes 3-4: 10 seconds;
Lanes 5-6: 32 seconds

Blocking buffer and concentration: 5% NFDM/TBST

Negative control: liver (lanes 2, 4 and 6, PMID: 10395956)

POMC is a glycoprotein and can be processed into 16kDa N-POMC. The molecular weight observed is consistent with what has been described in the literature (PMID 1814247, PMID 26880796, PMID 20639461).

Immunohistochemical analysis of paraffin-embedded human pituitary tissue labeling POMC with ab254257 at 1/4000 dilution at room temperature for 30 mins, followed by ready to use rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human pituitary is observed (PMID: 21622576). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was performed. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

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

Negative control: No staining on mouse spleen (PMID: 10395956)

Immunohistochemical analysis of paraffin-embedded mouse pituitary tissue labeling POMC with ab254257 at 1/4000 dilution at room temperature for 30 mins, followed by ready to use rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was performed. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of 0.2% Triton X-100 permeabilized, 4% PFA fixed mouse pituitary tissue labeling POMC with ab254257 at 1/500 dilution, followed by AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. DAPI was used as nuclear counterstain. Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) was performed. Positive staining on mouse pituitary (PMID: 18601691).

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

Negative control: No stain on liver (PMID: 14578285)

Immunohistochemical analysis of 0.2% Triton X-100 permeabilized, 4% PFA fixed mouse liver tissue labeling POMC with ab254257 at 1/100 dilution, followed by AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. DAPI was used as nuclear counterstain. Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) was performed. No staining on mouse liver.

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

Immunohistochemical analysis of paraffin-embedded rat pituitary tissue labeling POMC with ab254257 at 1/4000 dilution at room temperature for 30 mins, followed by ready to use rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat pituitary is observed (PMID: 21622576). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was performed. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

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

Immunohistochemical analysis of paraffin-embedded mouse pituitary tissue labeling POMC with ab254257 at 1/4000 dilution at room temperature for 30 mins, followed by ready to use rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse pituitary is observed (PMID: 21622576). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was performed. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

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

Negative control: No stain on liver (PMID: 14578285)

Immunohistochemical analysis of 0.2% Triton X-100 permeabilized, 4% PFA fixed rat liver tissue labeling POMC with ab254257 at 1/100 dilution, followed by AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. DAPI was used as nuclear counterstain. Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) was performed. No staining on rat liver.

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

Immunohistochemical analysis of 0.2% Triton X-100 permeabilized, 4% PFA fixed rat pituitary tissue labeling POMC with ab254257 at 1/500 dilution, followed by AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. DAPI was used as nuclear counterstain. Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) was performed. Positive staining on rat pituitary (PMID: 18601691).

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

Immunocytochemical analysis of 100% methanol fixed, 0.1% TritonX-100 permeabilised AtT-20 cells labeling POMC with ab254257 at 1/100 dilution. ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) was used as a secondary antibody at 1/1000 dilution. Counterstained with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution. Nuclear staining: DAPI.

Confocal image showing cytoplasmic staining in AtT-20 cell line