ATM Substrates Antibody Sampler Kit
cat.: HAK21187
Product Type: Antibody Sampler Kit
Species reactivity: Human
Applications: WB
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: O96017 Human | O96017 Human | Q9Z265 Mouse | P04637 Human | P04637 Human | Q92878 Human | P70388 Mouse | Q9JIL8 Rat | O60934 Human | Q9R207 Mouse | Q9JIL9 Rat | O60934 Human | Q9R207 Mouse
Entrez Gene: 114212 Rat
Images
HAK21187_1.jpg Fig1: Western blot analysis of Rad50 on different lysates with Rabbit anti-Rad50 antibody (HA721492) at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: K-562 cell lysate (20 µg/Lane)
Lane 3: U-87 MG cell lysate (20 µg/Lane)
Lane 4: U-937 cell lysate (20 µg/Lane)
Lane 5: Caco-2 cell lysate (20 µg/Lane)
Lane 6: Mouse testis tissue lysate (40 µg/Lane)
Lane 7: Rat testis tissue lysate (40 µg/Lane)

Predicted band size: 154 kDa
Observed band size: 154 kDa

Exposure time: 3 minutes;

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 (HA721492) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.
HAK21187_2.jpg Fig2: Western blot analysis of Chk2 on different lysates with Rabbit anti-Chk2 antibody (ET1610-52) at 1/1,000 dilution.

Lane 1: A549-si NT cell lysate
Lane 2: A549-si Chk2 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 61 kDa
Observed band size: 61 kDa

Exposure time: 40 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 (ET1610-52) 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.
HAK21187_3.jpg Fig3: Immunohistochemical analysis of paraffin-embedded human testis tissue with Rabbit anti-Chk2 antibody (ET1610-52) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (ET1610-52) 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.
HAK21187_4.jpg Fig4: Western blot analysis of Phospho-Chk2 (T68) on different lysates with Rabbit anti-Phospho-Chk2 (T68) antibody (HA721633) at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 20μM Etoposide for 2 hours cell lysate
Lane 3: Jurkat cell lysate
Lane 4: Jurkat treated with 25μM Etoposide for 5 hours cell lysate
Lane 5: NIH/3T3 cell lysate
Lane 6: NIH/3T3 treated with 25μM Etoposide for 5 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 61 kDa
Observed band size: 61 kDa

Exposure time: 5 minutes;
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 (HA721633) 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.
HAK21187_5.jpg Fig5: Chk2 was immunoprecipitated from 0.2 mg A549 cell lysate with ET1610-52 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1610-52 at 1/2,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: A549 cell lysate (input)
Lane 2: ET1610-52 IP in A549 cell lysate
Lane 3: Rabbit IgG instead of ET1610-52 in A549 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 10 seconds; ECL: K1801
HAK21187_6.jpg Fig6: Western blot analysis of p53 on different lysates with Rabbit anti-p53 antibody (ET1605-16) at 1/5,000 dilution.

Lane 1: HAP1-parental cell lysate
Lane 2: HAP1-p53 KD cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 53 kDa
Observed band size: 53 kDa

Exposure time: 2 minutes; 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 (ET1605-16) 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.
HAK21187_7.jpg Fig7: Immunocytochemistry analysis of A431 cells labeling p53 with Rabbit anti-p53 antibody (ET1605-16) 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-p53 antibody (ET1605-16) 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.
HAK21187_8.jpg Fig8: Western blot analysis of Phospho-p53 (S15) on different lysates with Rabbit anti-Phospho-p53 (S15) antibody (HA721756) at 1/1,000 dilution.

Lane 1: HT-29 cell lysate
Lane 2: HT-29 treated with 0.5μM Doxorubicin for 24 hours cell lysate
Lane 3: HT-29 treated with 0.5μM Doxorubicin for 24 hours, then treated with λpp for 1 hour cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 53 kDa
Observed band size: 53 kDa

Exposure time: 1 minute 46 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 (HA721756) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21187_9.jpg Fig9: Immunocytochemistry analysis of HT-29 treated with or without 0.5μM Doxorubicin for 24 hours cells labeling Phospho-p53 (S15) with Rabbit anti-Phospho-p53 (S15) antibody (HA721756) at 1/10,000 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-p53 (S15) antibody (HA721756) at 1/10,000 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.
HAK21187_10.jpg Fig10: Western blot analysis of p95 / NBS1 on different lysates with Rabbit anti-p95 / NBS1 antibody (ET1610-26) at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: NIH/3T3 cell lysate (20 µg/Lane)
Lane 3: PC-12 cell lysate (20 µg/Lane)
Lane 4: Mouse testis tissue lysate (40 µg/Lane)

Predicted band size: 85 kDa
Observed band size: 85 kDa

Exposure time: 3 minutes; 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 (ET1610-26) 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.
HAK21187_11.jpg Fig11: Immunohistochemical analysis of paraffin-embedded human large intestine tissue with Rabbit anti-p95 / NBS1 antibody (ET1610-26) at 1/100 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (ET1610-26) at 1/100 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.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.