ET7110-11

Phospho-KAP1 (S824) Recombinant Rabbit Monoclonal Antibody [JE50-99]

cat.: ET7110-11

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IHC-P, IP, Dot Blot, IF-Cell
Clonality:	Monoclonal
Clone number:	JE50-99

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 89 kDa
Isotype:	IgG
Immunogen:	Synthetic phospho-peptide corresponding to residues surrounding Ser824 of human KAP1.
Positive control:	HeLa treated with 20μM Etoposide for 2 hours cell lysate, HeLa treated with 100μM Etoposide for 4 hours cell lysate, C6 treated with 25μM Etoposide for 5 hours cell lysate, NIH/3T3 treated with 25μM Etoposide for 5 hours cell lysate, HeLa cells treated with 25μM Etoposide for 5 hours, NIH/3T3 cells treated with 25μM Etoposide for 5 hours, human tonsil tissue, human breast tissue, human gastric carcinoma tissue.
Subcellular location:	Nucleus.
Recommended Dilutions: WB IHC-P IP Dot Blot IF-Cell	1:500-1:2,000 1:50-1:200 1:10-1:50 Use at an assay dependent concentration. 1:100
Uniprot #:	SwissProt: Q13263 Human \| Q62318 Mouse \| O08629 Rat
Alternative names:	E3 SUMO protein ligase TRIM28 E3 SUMO-protein ligase TRIM28 FLJ29029 KAP 1 KAP-1 KRAB associated protein 1 KRAB interacting protein 1 KRAB-associated protein 1 KRAB-interacting protein 1 KRIP 1 KRIP-1 KRIP1 Nuclear corepressor KAP 1 Nuclear corepressor KAP-1 RING finger protein 96 RNF96 TF1B TIF1 beta TIF1-beta TIF1B TIF1B_HUMAN Transcription intermediary factor 1 beta Transcription intermediary factor 1-beta Trim28 Tripartite motif containing 28 tripartite motif containing protein 28 Tripartite motif-containing protein 28

Images

Fig1: Western blot analysis of Phospho-KAP1 (S824) on different lysates with Rabbit anti-Phospho-KAP1 (S824) antibody (ET7110-11) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 20μM Etoposide for 2 hours cell lysate
Lane 3: HeLa cell lysate
Lane 4: HeLa treated with 100μM Etoposide for 4 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 89 kDa
Observed band size: 110 kDa

Exposure time: 1 minute 58 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET7110-11) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig2: Western blot analysis of Phospho-KAP1 (S824) on different lysates with Rabbit anti-Phospho-KAP1 (S824) antibody (ET7110-11) at 1/1,000 dilution.

Lane 1: C6 cell lysate
Lane 2: C6 treated with 25μM Etoposide for 5 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 89 kDa
Observed band size: 110 kDa

Exposure time: 1 minute;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET7110-11) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig3: Western blot analysis of Phospho-KAP1 (S824) on different lysates with Rabbit anti-Phospho-KAP1 (S824) antibody (ET7110-11) at 1/1,000 dilution.

Lane 1: NIH/3T3 cell lysate
Lane 2: NIH/3T3 treated with 25μM Etoposide for 5 hours cell lysate
Lane 3: NIH/3T3 treated with 25μM Etoposide for 5 hours cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 89 kDa
Observed band size: 110 kDa

Exposure time: 1 minute 7 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET7110-11) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig4: Immunocytochemistry analysis of HeLa cells treated with or without 25μM Etoposide for 5 hours labeling Phospho-KAP1 (S824) with Rabbit anti-Phospho-KAP1 (S824) antibody (ET7110-11) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-KAP1 (S824) antibody (ET7110-11) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

	Fig5: Immunocytochemistry analysis of NIH/3T3 cells treated with or without 25μM Etoposide for 5 hours labeling Phospho-KAP1 (S824) with Rabbit anti-Phospho-KAP1 (S824) antibody (ET7110-11) at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-KAP1 (S824) antibody (ET7110-11) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
	Fig6: Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Phospho-KAP1 (S824) antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET7110-11, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig7: Immunohistochemical analysis of paraffin-embedded human breast tissue using anti-Phospho-KAP1 (S824) antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET7110-11, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Fig8: Immunohistochemical analysis of paraffin-embedded human gastric carcinoma tissue using anti-Phospho-KAP1 (S824) antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET7110-11, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.