Anti-KDM1/LSD1 antibody - Nuclear Marker

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM1/LSD1 antibody - Nuclear Marker (AB37165)

IHC image of KDM1/LSD1 staining in human prostate formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab37165, 1μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM1/LSD1 antibody - Nuclear Marker (AB37165)

Immunohistochemistry (Formalin-fixed paraffin embedded sections) of human breast carcinoma tissue labeling KDM1/LSD1 with ab37165 at 2μg/ml.

• WB

Supplier Data

Western blot - Anti-KDM1/LSD1 antibody - Nuclear Marker (AB37165)

Diluting buffer and concentration : 1 hour at room temperature in 5% NFDM/TBST.

All lanes:

Western blot - Anti-KDM1/LSD1 antibody - Nuclear Marker (ab37165) at 0.2 µg/mL

Lane 1:

HEK-293 (human epithelial cell line from embryonic kidney) cell lysate at 15 µg

Lane 2:

A431 (human epidermoid carcinoma cell line) cell lysate at 15 µg

Lane 3:

A549 (human lung carcinoma cell line) cell lysate at 15 µg

Lane 4:

Caco-2 (human colorectal adenocarcinoma cell line) cell lysate at 15 µg

Lane 5:

Daudi (human Burkitt's lymphoma cell line) cell lysate at 15 µg

Lane 6:

HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 15 µg

Lane 7:

HepG2 (human liver hepatocellular carcinoma cell line) cell lysate at 15 µg

Lane 8:

K562 (human chronic myelogenous leukemia cell line from bone marrow) cell lysate at 15 µg

Lane 9:

MCF7 (human breast adenocarcinoma cell line) cell lysate at 15 µg

Lane 10:

Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate at 15 µg

Lane 11:

SK-N-SH (human neuroblastoma cell line) cell lysate at 15 µg

Lane 12:

THP-1 (human monocytic leukemia cell line) cell lysate at 15 µg

Lane 13:

NIH/3T3 (mouse embryo fibroblast cell line) cell lysate at 15 µg

Lane 14:

YB2/0 (rat spleen lymphoblast cell line) cell lysate at 15 µg

Secondary

All lanes:

Goat anti-rabbit IgG (HRP) at 1/10000 dilution

Predicted band size: 92 kDa

Observed band size: 110 kDa

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• WB

Unknown

Western blot - Anti-KDM1/LSD1 antibody - Nuclear Marker (AB37165)

Lane 1:

Western blot - Anti-KDM1/LSD1 antibody - Nuclear Marker (ab37165) at 1 µg/mL

Lane 2:

Western blot - Anti-KDM1/LSD1 antibody - Nuclear Marker (ab37165) at 2 µg/mL

All lanes:

Mouse P815 cell lysate at 15 µg

Predicted band size: 92 kDa

Observed band size: 105 kDa

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• WB

Supplier Data

Western blot - Anti-KDM1/LSD1 antibody - Nuclear Marker (AB37165)

Diluting buffer and concentration : 1 hour at room temperature in 5% NFDM/TBST.

All lanes:

Western blot - Anti-KDM1/LSD1 antibody - Nuclear Marker (ab37165) at 0.2 µg/mL

Lane 1:

Mouse Testis tissue lysate at 15 µg

Lane 2:

Mouse Spleen tissue lysate at 15 µg

Lane 3:

Mouse Kidney tissue lysate at 15 µg

Lane 4:

Mouse Colon tissue lysate at 15 µg

Lane 5:

Mouse Stomach tissue lysate at 15 µg

Lane 6:

Mouse Brain tissue lysate at 15 µg

Lane 7:

Mouse Skin tissue lysate at 15 µg

Lane 8:

Mouse Skeletal Muscle tissue lysate at 15 µg

Lane 9:

Mouse Bladder tissue lysate at 15 µg

Secondary

All lanes:

Goat anti-rabbit IgG (HRP) at 1/10000 dilution

Predicted band size: 92 kDa

Observed band size: 110 kDa

false

• WB

CiteAb

Western blot - Anti-KDM1/LSD1 antibody - Nuclear Marker (AB37165)

KDM1/LSD1 western blot using anti-KDM1/LSD1 antibody - Nuclear Marker ab37165. Publication image and figure legend from Storm, M. P., Kumpfmueller, B., et al., 2014, PLoS One, PubMed 24594919.

ab37165 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab37165 please see the product overview.

Zscan4 acts as a transcriptional regulator and interacts with LSD-1 and CtBP2.A. Schematic showing the structure of GAL4-DNA binding domain fusions with Zscan4c. B. Plasmids expressing the GAL-4-DNA binding domain fusions indicated were co-transfected into HEK293 cells along with pGL4-GAL4-UAS plasmid and the pRenilla plasmid (to assess transfection efficiency). 48 h after transfection luciferase activity was determined using Dual-Glo system, according to the manufacturer's recommendations (Promega). Mean and SEM of 3 independent experiments are shown. *, p<0.05, **, p<0.005 following an ANOVA and Tukey's post-hoc test. C. ESCs engineered to express an N-Terminal eGFP-Zscan4c fusion protein under the control of the pTRE tight Dox-inducible expression system were cultured in the absence (-) or presence (+) of Dox for 72 h. Nuclear extracts were prepared (Nuc) and immunoprecipitates prepared from 80 μg of protein per sample using either GFP-Trap-A beads or anti-LSD-1 antibodies. Precipitates were divided into two aliquots and separated through 10% polyacrylamide on duplicate gels, prior to immunoblot. Immunoblot was performed with anti-GFP, anti-CtBP2 or anti-Zscan4 antibodies, sequentially with one immunoblot. The duplicate immunoblot was probed with anti-LSD-1 antibodies. Positions of precipitated proteins are indicated. D. ESCs engineered to express a C-Terminally V5 epitope-tagged version of Zscan4c under the control of the Tet-off expression system were cultured in the presence (+) or absence (-) of Tet for 72 h. Nuclear extracts were made (Nuc) and immunoprecipitates prepared from 80 μg of protein per sample using either anti-LSD-1 or anti-CtBP2 antibodies or protein-A sepharose (PAS) alone as a control. Precipitates were separated through 7.5% polyacrylamide gels prior to immunoblot. Immunoblot was first performed with anti-Zscan4 antibodies. The blots were then stripped and reprobed with anti-V5 epitope antibodies. Positions of precipitated proteins are indicated. E. Nuclear extracts (Nuc) were prepared from E14 ESCs and precipitates from 200 μg of protein per sample generated using anti-Zscan4, anti-LSD-1 or anti-CtBP2 antibodies. Precipitates were separated through a 6.5% polyacrylamide gel prior to immunoblot and the lanes between the samples were left blank to avoid the potential of any bleed-through. Immunoblot was performed with anti-Zscan4 antibodies. Positions of precipitated proteins are indicated. F. ESCs engineered to express a C-Terminally V5 epitope-tagged version of Zscan4c under the control of the Tet-off expression system were cultured in the presence (+) or absence (-) of Tet for 72 h. Nuclear extracts were prepared (Nuc) and immunoprecipitates prepared from 80 μg of protein per sample using anti-V5 epitope antibodies. Precipitates were separated through a 7.5% polyacrylamide gel prior to immunoblot. Immunoblot was performed with anti-Zscan4 antibodies. Positions of precipitated proteins are indicated.

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