Disulfidptosis Antibody Sampler Kit
cat.: HAK21206
Product Type: Antibody Sampler Kit
Species reactivity: Human, Mouse, Rat
Applications: WB, IHC-P, IF-Cell
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
Uniprot #: SwissProt: Q9UPY5 Human | Q9WTR6 Mouse | P21333 Human | Q8BTM8 Mouse | P11166 Human | P17809 Mouse | P11167 Rat | P52789 Human | O08528 Mouse | P27881 Rat | P00338 Human | P06151 Mouse | P04642 Rat | P35579 Human | Q8VDD5 Mouse | Q62812 Rat | P07237 Human | P09103 Mouse | P04785 Rat | Q9Y490 Human | P26039 Mouse
Entrez Gene: 310392 Rat | 313494 Rat
Gene ID: 293860 Rat
HAK21206 - Page 2
Images
HAK21206_1.jpg Fig1: Western blot analysis of xCT / SLC7A11 on different lysates with Rabbit anti-xCT / SLC7A11 antibody (HA724114) at 1/2,000 dilution.

Lane 1: A549 cell lysate (no heat) (20 µg/Lane)
Lane 2: Mouse cerebellum tissue lysate (no heat) (20 µg/Lane)
Lane 3: Rat brain tissue lysate (no heat) (20 µg/Lane)

Notice: no heat means the lysate is not boiled.

Exposure time: 25 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 (HA724114) at 1/2,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21206_2.jpg Fig2: Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-xCT / SLC7A11 antibody (HA724114) at 1/50 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 (HA724114) at 1/50 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.
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HAK21206_3.jpg Fig3: Immunocytochemistry analysis of A549 cells labeling xCT / SLC7A11 with Rabbit anti-xCT / SLC7A11 antibody (HA724114) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-xCT / SLC7A11 antibody (HA724114) 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 (HA601187 , 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.
HAK21206_4.jpg Fig4: Flow cytometric analysis of A549 cells labeling xCT / SLC7A11.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA724114, 1μg/mL) (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).
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HAK21206_5.jpg Fig5: Western blot analysis of Filamin A on different lysates with Rabbit anti-Filamin A antibody (ET1601-3) at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HEK-293 cell lysate
Lane 3: A549 cell lysate
Lane 4: NIH/3T3 cell lysate
Lane 5: C2C12 cell lysate
Lane 6: C6 cell lysate
Lane 7: COS-1 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 281 kDa
Observed band size: 281 kDa

Exposure time: 3 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 (ET1601-3) 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.
HAK21206_6.jpg Fig6: Immunocytochemistry analysis of HeLa cells labeling Filamin A with Rabbit anti-Filamin A antibody (ET1601-3) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-Filamin A antibody (ET1601-3) at 1/200 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 (HA601187, 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.
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HAK21206_7.jpg Fig7: Immunocytochemistry analysis of C6 cells labeling Filamin A with Rabbit anti-Filamin A antibody (ET1601-3) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-Filamin A antibody (ET1601-3) at 1/200 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 (HA601187, 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.
HAK21206_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded mouse uterus tissue using anti-Filamin A antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) 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 (ET1601-3, 1/400) 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.
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HAK21206_9.jpg Fig9: Western blot analysis of Glucose Transporter GLUT1 on different lysates with Rabbit anti-Glucose Transporter GLUT1 antibody (ET1601-10) at 1/50,000 dilution and competitor's antibody at 1/50,000 dilution.

Lane 1: HeLa cell lysate (no heat) (20 µg/Lane)
Lane 2: HT-29 cell lysate (no heat) (20 µg/Lane)
Lane 3: HepG2 cell lysate (no heat) (20 µg/Lane)
Lane 4: NIH/3T3 cell lysate (no heat) (20 µg/Lane)
Lane 5: L-929 cell lysate (no heat) (20 µg/Lane)
Lane 6: Mouse brain tissue lysate (no heat) (20 µg/Lane)
Lane 7: Rat brain tissue lysate (no heat) (20 µg/Lane)

Notice: no heat means the lysate is not boiled.

Predicted band size: 54 kDa
Observed band size: 45-60 kDa
Exposure time: Lane 1-7 (left): 20 seconds; Lane 1-7 (right): 1 minute 30 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 (ET1601-10) at 1/50,000 dilution and competitor's antibody at 1/50,000 dilution were 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.
HAK21206_10.jpg Fig10: Immunocytochemistry analysis of HeLa cells labeling Glucose Transporter GLUT1 with Rabbit anti-Glucose Transporter GLUT1 antibody (ET1601-10) at 1/500 dilution and competitor's antibody at 1/200 dilution.

Cells were fixed in 100% precooled methanol 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-Glucose Transporter GLUT1 antibody (ET1601-10) at 1/500 dilution and competitor's antibody at 1/200 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.
HAK21206 - Page 7
HAK21206_11.jpg Fig11: Immunocytochemistry analysis of C6 cells labeling Glucose Transporter GLUT1 with Rabbit anti-Glucose Transporter GLUT1 antibody (ET1601-10) at 1/500 dilution.

Cells were fixed in 100% precooled methanol 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-Glucose Transporter GLUT1 antibody (ET1601-10) at 1/500 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.
HAK21206_12.jpg Fig12: Flow cytometric analysis of Jurkat cells labeling Glucose Transporter GLUT1.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1601-10, red) at 1/1,000 dilution and competitor's antibody (red) at 1/50 dilution, compared with Rabbit IgG Isotype Control (blue). 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).
HAK21206 - Page 8
HAK21206_13.jpg Fig13: Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-Glucose Transporter GLUT1 antibody (ET1601-10) at 1/5,000 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 (ET1601-10) 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.
HAK21206_14.jpg Fig14: Application: IF-tissue

Species: Human

Site: Placenta

Sample: Paraffin-embedded section

Antibody concentration: 1/500
HAK21206 - Page 9
HAK21206_15.jpg Fig15: Application: Immunofluorescence (IHC-Fr)

Species: Mouse

Site: Cerebral cortex

Sample: Frozen section

Antibody concentration: 1/1,000

Antigen retrieval: Not required
HAK21206_16.jpg Fig16: Western blot analysis of Hexokinase II on different lysates with Rabbit anti-Hexokinase II antibody (HA722933) at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HCT 116 cell lysate (20 µg/Lane)
Lane 3: NIH/3T3 cell lysate (20 µg/Lane)
Lane 4: PC-12 cell lysate (20 µg/Lane)
Lane 5: COS-1 cell lysate (20 µg/Lane)
Lane 6: Mouse skeletal muscle tissue lysate (40 µg/Lane)
Lane 7: Mouse testis tissue lysate (40 µg/Lane)
Lane 8: Rat skeletal muscle tissue lysate (40 µg/Lane)
Lane 9: Rat testis tissue lysate (40 µg/Lane)

Predicted band size: 102 kDa
Observed band size: 102 kDa

Exposure time: 8 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 (HA722933) 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.
HAK21206 - Page 10
HAK21206_17.jpg Fig17: Immunocytochemistry analysis of NIH/3T3 cells labeling Hexokinase II with Rabbit anti-Hexokinase II antibody (HA722933) at 1/50 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-Hexokinase II antibody (HA722933) at 1/50 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 (HA601187, 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.
HAK21206_18.jpg Fig18: Hexokinase II was immunoprecipitated from 0.2 mg HeLa cell lysate with HA722933 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722933 at 1/2,000 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: HA722933 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of HA722933 in HeLa cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 6 seconds; ECL: K1801
HAK21206 - Page 11
HAK21206_19.jpg Fig19: Western blot analysis of LDHA on different lysates with Rabbit anti-LDHA antibody (HA723547) at 1/5,000 dilution.

Lane 1: HepG2 cell lysate (20 µg/Lane)
Lane 2: HeLa cell lysate (20 µg/Lane)
Lane 3: MCF7 cell lysate (20 µg/Lane)
Lane 4: A431 cell lysate (20 µg/Lane)
Lane 5: 293T cell lysate (20 µg/Lane)
Lane 6: PANC-1 cell lysate (20 µg/Lane)
Lane 7: U-87 MG cell lysate (20 µg/Lane)
Lane 8: MDA-MB-231 cell lysate (20 µg/Lane)
Lane 9: NIH/3T3 cell lysate (20 µg/Lane)
Lane 10: RAW264.7 cell lysate (20 µg/Lane)
Lane 11: Neuro-2a cell lysate (20 µg/Lane)
Lane 12: C2C12 cell lysate (20 µg/Lane)
Lane 13: C6 cell lysate (20 µg/Lane)
Lane 14: PC-12 cell lysate (20 µg/Lane)

Predicted band size: 37 kDa
Observed band size: 33 kDa
Exposure time: 6 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 (HA723547) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21206_20.jpg Fig20: Immunocytochemistry analysis of C2C12 cells labeling LDHA with Rabbit anti-LDHA antibody (HA723547) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-LDHA antibody (HA723547) at 1/500 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 (HA601187, 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.
HAK21206 - Page 12
HAK21206_21.jpg Fig21: Western blot analysis of non-muscle Myosin IIA on different lysates with Rabbit anti-non-muscle Myosin IIA antibody (HA723600) at 1/5,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HUVEC cell lysate (20 µg/Lane)
Lane 3: A431 cell lysate (20 µg/Lane)
Lane 4: HT-29 cell lysate (20 µg/Lane)
Lane 5: NIH/3T3 cell lysate (20 µg/Lane)
Lane 6: C2C12 cell lysate (20 µg/Lane)
Lane 7: PC-12 cell lysate (20 µg/Lane)
Lane 8: A549 cell lysate (20 µg/Lane)
Lane 9: Mouse kidney tissue lysate (40 µg/Lane)
Lane 10: Mouse lung tissue lysate (40 µg/Lane)
Lane 11: Mouse liver tissue lysate (40 µg/Lane)
Lane 12: Rat kidney tissue lysate (40 µg/Lane)
Lane 13: Rat lung tissue lysate (40 µg/Lane)
Lane 14: Rat liver tissue lysate (40 µg/Lane)

Predicted band size: 227 kDa
Observed band size: 227 kDa
Exposure time: 1 minute 30 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 (HA723600) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21206_22.jpg Fig22: Immunocytochemistry analysis of HeLa cells labeling non-muscle Myosin IIA with Rabbit anti-non-muscle Myosin IIA antibody (HA723600) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-non-muscle Myosin IIA antibody (HA723600) 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 (HA601187, 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.
HAK21206 - Page 13
HAK21206_23.jpg Fig23: Western blot analysis of P4HB on different lysates with Rabbit anti-P4HB antibody (ET7110-92) at 1/2,000 dilution.

Lane 1: THP-1 cell lysate (20 µg/Lane)
Lane 2: HepG2 cell lysate (20 µg/Lane)
Lane 3: MCF7 cell lysate (20 µg/Lane)
Lane 4: NIH/3T3 cell lysate (20 µg/Lane)
Lane 5: COS-1 cell lysate (20 µg/Lane)
Lane 6: Mouse liver tissue lysate (40 µg/Lane)
Lane 7: Rat liver tissue lysate (40 µg/Lane)

Predicted band size: 57 kDa
Observed band size: 57 kDa

Exposure time: 6 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 (ET7110-92) 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.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.