HAK21176

Non-Homologous End Joining Antibody Sampler Kit

cat.: HAK21176

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: P16104 Human \| P27661 Mouse \| D3ZXP3 Rat \| P16104 Human \| P27661 Mouse \| P49917 Human \| P12956 Human \| P23475 mouse \| P13010 Human Entrez Gene: 25019 Rat Unigene: 2850 Rat

Images

Fig1: Western blot analysis of Histone H2A.X on different lysates with Rabbit anti-Histone H2A.X antibody (ET1705-97) at 1/1,000 dilution.

Lane 1: Raji cell lysate
Lane 2: NIH/3T3 cell lysate
Lane 3: C6 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 15 kDa
Observed band size: 15 kDa

Exposure time: 3 minutes;

15% 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 (ET1705-97) 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: Immunofluorescence staining of paraffin- embedded mouse testis tissue using anti-Histone H2A.X antibody.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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with ET1705-97 at 1/50 dilution for 10 hours at 4℃ and detected using Alexa Fluor® 488 conjugate-Goat anti-Rabbit IgG (H+L) Secondary Antibody at a dilution of 1:500 for 1 hour at room temperature.

Fig3: Immunohistochemical analysis of paraffin-embedded mouse testis tissue with Rabbit anti-Histone H2A.X antibody (ET1705-97) 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 (ET1705-97) 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.

Fig4: Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2,000 dilution.

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

Lysates/proteins at 20 µg/Lane.

Predicted band size: 15 kDa
Observed band size: 15/20 kDa

Exposure time: 53 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 (ET1602-2) at 1/2,000 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.

Fig5: Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/5,000 dilution.

Lane 1: NIH/3T3 cell lysate (20 µg/Lane)
Lane 2: NIH/3T3 treated with 25μM Etoposide for 5 hours cell lysate (20 µg/Lane)

Predicted band size: 15 kDa
Observed band size: 15 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 (ET1602-2) at 1/5,000 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.

Fig6: Immunocytochemistry analysis of HeLa cells untreated / treated with 20μM etoposide for 2 hours labeling Phospho-Histone H2A.X (S139) with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2,000 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-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2,000 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 (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.

Fig7: Application: IF-tissue

Species: Cynomolgus monkey

Site: Lung

Sample: Paraffin-embedded section

Antibody concentration: 1/500

Fig8: Western blot analysis of Ku80 on different lysates with Rabbit anti-Ku80 antibody (ET1610-40) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: MCF7 cell lysate
Lane 3: A549 cell lysate
Lane 4: COS-1 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 83 kDa
Observed band size: 83 kDa

Exposure time: 10 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 (ET1610-40) 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.

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