Anti-REG3A + REG3G antibody [EPRR18188]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-REG3A + REG3G antibody [EPRR18188] (AB202057)

Flow cytometry (intracellular) analysis of transfected 293T cells. Cells were fixed with 4% PFA and permeabilised with 90% methanol before staining for REG3G and REG3A in 293T cells (human embryonic kidney epithelial cell) transfected with a human REG3G expression vector containing a Myc-His-tag^® (Right), and 293T cells transfected with a human REG3A expression vector containing a Myc-His-tag^® (Middle). Primary antibody ab202057 used at a 1/5000 dilution (0.01μg). Staining was compared with 293T cells transfected with an empty expression vector containing a myc-His-tag^® (Left). Cells were co-stained with Myc tag conjugated to Alexa Fluor^®647. Goat anti‑rabbit IgG (Alexa Fluor^® 488, ab150081) was used as the secondary antibody at a 1/5000 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-REG3A + REG3G antibody [EPRR18188] (AB202057)

Flow cytometric overlay histogram of 4% paraformaldehyde-fixed, 90% methanol permeabilised HT-29 (Human colorectal adenocarcinoma epithelial cell) cells labelling REG3A + REG3G with Anti-REG3A + REG3G antibody (ab202057) at 1 µg/test (1/50 dilution) (red line). Isotype control antibody (black line) was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody. A Goat Anti-Rabbit IgG Alexa Fluor^® 488 secondary antibody (ab150081) was used at 1/5000 dilution. Unlabelled sample (blue line), consisting of cells without incubation with either the primary or secondary antibody, was also used as a control.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-REG3A + REG3G antibody [EPRR18188] (AB202057)

Immunofluorescent analysis of 100% Methanol-fixed 0.1% Triton X-100 permeabilized HT-29 (Human colorectal adenocarcinoma cells) cells labeling REG3G + REG3A with ab202057 at 1/1000 dilution followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic staining on HT-29 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows :
-ve control 1 : ab202057 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-REG3A + REG3G antibody [EPRR18188] (AB202057)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% Triton X-100 permeabilized BxPC-3 (Human pancreas adenocarcinoma cells) cells labeling REG3G + REG3A with ab202057 at 1/1000 dilution followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic staining on BxPC-3 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows :
-ve control 1 : ab202057 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-REG3A + REG3G antibody [EPRR18188] (AB202057)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling REG3G + REG3A with ab202057 at 1/600 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic staining on Human pancreas tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-REG3A + REG3G antibody [EPRR18188] (AB202057)

Blocking/Dilution buffer : 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literatures PMID : 12890698 & 22943287 for REG3A and PMID : 22252863 & 21254165 for REG3G.
Please note both Human fetal liver and brain represent negative controls for REG3A and REG3G.

All lanes:

Western blot - Anti-REG3A + REG3G antibody [EPRR18188] (ab202057) at 1/1000 dilution

Lane 1:

Human small intestine tissue lysate at 10 µg

Lane 2:

Human fetal liver tissue lysate at 10 µg

Lane 3:

Human fetal brain tissue lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 19 kDa,35 kDa,66 kDa

Observed band size: 16 kDa,39 kDa,67 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-REG3A + REG3G antibody [EPRR18188] (AB202057)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-REG3A + REG3G antibody [EPRR18188] (ab202057) at 1/10000 dilution

Lane 1:

REG3A recombinant protein fragment at 0.01 µg

Lane 2:

REG3G recombinant protein fragment at 0.01 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 19 kDa

Observed band size: 16 kDa

false

Exposure time: 15s