ET7107-17

OGT Recombinant Rabbit Monoclonal Antibody [JB44-39]

cat.: ET7107-17

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IHC-P, FC, IP
Clonality:	Monoclonal
Clone number:	JB44-39

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. 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: 117 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within Human OGT aa 997-1,046 / 1,046.
Positive control:	HeLa cell lysate, MCF7 cell lysate, HEK-293 cell lysate, NIH/3T3 cell lysate, PC-12 cell lysate, Mouse brain tissue lysate, Rat brain tissue lysate, HeLa, MCF7, PC-12, human testis tissue, human liver tissue, mouse testis tissue, mouse epididymis tissue, rat testis tissue.
Subcellular location:	Nucleus. Cytoplasm. Membrane.
Recommended Dilutions: WB IF-Cell IHC-P FC IP	1:2,000 1:100 1:200-1:1,000 1:1,000 1-2μg/sample
Uniprot #:	SwissProt: O15294 Human \| Q8CGY8 Mouse \| P56558 Rat
Alternative names:	FLJ23071 GlcNAc transferase HRNT1 MGC22921 O GlcNAc O GlcNAc transferase p110 subunit O GlcNAc transferase subunit p110 O linked N acetylglucosamine (GlcNAc) transferase (UDP N acetylglucosamine:polypeptide N acetylglucosaminyl transferase) O linked N acetylglucosamine (GlcNAc) transferase O linked N acetylglucosamine transferase 110 kDa subunit O-GlcNAc transferase subunit p110 O-linked N-acetylglucosamine transferase 110 kDa subunit ogt OGT1_HUMAN UDP N acetylglucosamine peptide N acetylglucosaminyltransferase 110 kDa subunit UDP N acetylglucosamine peptide N acetylglucosaminyltransferase GlcNAc transferase UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase Uridinediphospho N acetylglucosamine:polypeptide beta N acetylglucosaminyl transferase

Images

Fig1: Western blot analysis of OGT on different lysates with Rabbit anti-OGT antibody (ET7107-17) at 1/2,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: MCF7 cell lysate (20 µg/Lane)
Lane 3: HEK-293 cell lysate (20 µg/Lane)
Lane 4: NIH/3T3 cell lysate (20 µg/Lane)
Lane 5: PC-12 cell lysate (20 µg/Lane)
Lane 6: Mouse brain tissue lysate (40 µg/Lane)
Lane 7: Rat brain tissue lysate (40 µg/Lane)

Predicted band size: 117 kDa
Observed band size: 117kDa

Exposure time: 46 seconds; ECL: K1801;

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 (ET7107-17) at 1/2,000 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: Immunocytochemistry analysis of HeLa cells labeling OGT with Rabbit anti-OGT antibody (ET7107-17) 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-OGT antibody (ET7107-17) 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.

Fig3: Immunocytochemistry analysis of MCF7 cells labeling OGT with Rabbit anti-OGT antibody (ET7107-17) 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-OGT antibody (ET7107-17) 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.

Fig4: Immunocytochemistry analysis of PC-12 cells labeling OGT with Rabbit anti-OGT antibody (ET7107-17) 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-OGT antibody (ET7107-17) 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: Immunohistochemical analysis of paraffin-embedded human testis tissue with Rabbit anti-OGT antibody (ET7107-17) at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET7107-17) at 1/200 dilution for 1 hour 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.
	Fig6: Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-OGT antibody (ET7107-17) at 1/1,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET7107-17) at 1/1,000 dilution for 1 hour 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 mouse testis tissue with Rabbit anti-OGT antibody (ET7107-17) at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET7107-17) at 1/200 dilution for 1 hour 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 mouse epididymis tissue with Rabbit anti-OGT antibody (ET7107-17) at 1/1,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET7107-17) at 1/1,000 dilution for 1 hour 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.
	Fig9: Immunohistochemical analysis of paraffin-embedded rat testis tissue with Rabbit anti-OGT antibody (ET7107-17) at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET7107-17) at 1/200 dilution for 1 hour 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.
	Fig10: Flow cytometric analysis of HeLa cells labeling OGT. Cells were fixed and permeabilized. Then stained with the primary antibody (ET7107-17, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
	Fig11: Flow cytometric analysis of MCF7 cells labeling OGT. Cells were fixed and permeabilized. Then stained with the primary antibody (ET7107-17, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Fig12: OGT was immunoprecipitated from 0.2 mg HeLa cell lysate with ET7107-17 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using ET7107-17 at 1/1,000 dilution. Mouse Anti-Rabbit IgG kappa light chain secondary antibody (M1208-2) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: ET7107-17 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of ET7107-17 in HeLa cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 59 seconds; ECL: K1801

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