MA5-12933

antibody from Invitrogen Antibodies

Targeting: XRCC5 KARP-1, KU80, Ku86, KUB2

Provider product page for MA5-12933

Western blot

Immunocytochemistry

Immunoprecipitation

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [72]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [3]
Flow cytometry [3]
Other assay [9]

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Product number: MA5-12933 - Provider product page
Provider: Invitrogen Antibodies
Product name: Ku80 Monoclonal Antibody (111)
Antibody type: Monoclonal
Antigen: Other
Description: MA5-12933 targets Ku (p80) in FACS, ICC/IF, IP, and WB applications and shows reactivity with Human, Mouse and Non-human primate samples. This antibody does not cross-react with mouse in immunofluorescent applications, but does react with mouse in FACS applications. The MA5-12933 immunogen is human B cell nuclei from plasmacytoid 2p68 cells.
Reactivity: Human, Mouse
Host: Mouse
Isotype: IgG
Antibody clone number: 111
Vial size: 500 µL
Concentration: 0.2 mg/mL
Storage: 4° C

XRCC5 protein structure - MA5-12933 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of Ku (P80) using Ku (P80) Monoclonal Antibody (Product # MA5-12933) on HeLa Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Ku80 was achieved by transfecting HeLa with Ku80 specific siRNAs (Silencer® select Product # s14952, s14953). Western blot analysis (Fig. a) was performed using whole cell extracts from the Ku80 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Ku80 Monoclonal Antibody (111) (Product # MA5-12933, 0.4 ug/ml concentration) and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 0.25ug/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Ku80.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Ku80 Monoclonal Antibody (111), (Product # MA5-12933) and a 80 kDa band corresponding to Ku80 was observed in the cell lines tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of HeLa (Lane 1), MCF7 (Lane 2), K-562 (Lane 3), Jurkat (Lane 4), COS-7 (Lane 5) and A549 (Lane 6) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:500 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Ku (p80) (green) showing staining in the nucleus of HeLa cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Ku (p80) monoclonal antibody (Product # MA5-12933) in 3% BSA-PBS at a dilution of 1:150 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 100x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Ku (p80) (green) showing staining in the nucleus of MCF-7 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Ku (p80) monoclonal antibody (Product # MA5-12933) in 3% BSA-PBS at a dilution of 1:150 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 100x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Ku80 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Ku80 Monoclonal Antibody (111) (Product # MA5-12933) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing staining in nucleus. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Ku (p80) in HepG2 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Ku (p80) monoclonal antibody (Product # MA5-12933) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of Ku (p80) in MCF-7 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Ku (p80) monoclonal antibody (Product # MA5-12933) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of Ku (p80) in NIH-3T3 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Ku (p80) monoclonal antibody (Product # MA5-12933) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Ku (P80) using Ku (P80) Monoclonal Antibody (Product # MA5-12933) on Native Human T47D Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of Ku (P80) using Ku (P80) Monoclonal Antibody (Product # MA5-12933) on denatured Human BT474 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. LINP1 associates with NHEJ pathway proteins Ku80 and DNA-PKcs in Hela S3 cells a. Immunoprecipitation efficiency of Ku80 and DNA-PKcs specific antibodies detected by western blots. b and c. Results from RNA-IP. QRT-PCR assays showing the relative level of LINP1 in the complex immunoprecipitated by Ku80- and DNA-PKcs-specific antibodies. d. QRT-PCR assays showing the enrichment of LINP1 with specific biotinylated oligos. e. Western blots showing the Ku80 and DNA-PKcs protein levels in the complexes pulled down by LINP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 LINP1 RNA associates with Ku80 and DNA-PKcs ( a ) A schematic representation of the RNA pull-down assay. ( b ) Western analysis of Ku80 and DNA-PKcs levels in the protein complexes pulled down by either LINP1, antisense LINP1, or empty beads from whole cell extracts of MDA-MB-231 cells. ""Input"" refers to 5% input of whole cell lysates. ( c ) A schematic representation of CHART assay. ( d ) A schematic diagram of LINP1 RNA, the eleven C-oligos (red bars) designed for CHART assay, and the three primer sets (convergent arrows) used in the RNase-H sensitivity assay. RNase-H sensitivity was indicated with blocks with different shades of red, with darker shade indicating higher sensitivity. ( e ) Enrichment of LINP1 with C-oligo 1.2 and 1.4, as measured by qRT-PCR. GAPDH was used as negative control. ( f ) Presence of Ku80 and DNA-PKcs in C-oligo 1.2 and 1.4 enriched complexes, detected by Western analysis. C-oligo 1.2S and 1.4S were used as non-specific controls. ( g ) A schematic representation of an RNA-immunoprecipitation (RNA-IP) assay. ( h ) Results from native RNA-IP (upper panel) and CLIP (lower panel) with Ku80, Ku70, and DNA-PKcs specific antibodies. Western blots showing the immunoprecipitation efficiency of Ku80, Ku70 and DNA-PKcs. ( i ) Results from native RNA-IP (upper panel) and CLIP (lower panel) with Ku80, Ku70, and DNA-PKcs specific antibodies. qRT-PCR assays showing the level of LINP1 and GAPDH (non-specific control) in the co-precipitates. Ku80 (left)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: LINP1 serves as a modular scaffold in the NHEJ pathway (a) IR-induced DNA damage in control and LINP1 knockdown MDA-MB-231 cells, measured by the comet assay. Scale bar indicates 10 µm. (b) Levels of IR-induced DNA damage, quantified by the tail moment in the comet assay. Two-tailed Student’s t-test; * indicates p-value

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 XRCC4 is required for the recruitment of RIG-I to DSB sites. a HEK293T cells were transfected with empty vector or Flag-RIG-I. The cells were then lysed and immunoprecipitated with anti-Flag agarose beads. The beads were boiled and probed with indicated antibodies. b A549 cells were treated with IR (10 Gy, 1-2 h). Cells were lysed, and nuclear fractions were immunoprecipitated with anti-RIG-I antibody. The beads were treated with RNase A, boiled and blotted with indicated antibodies. c Control and XRCC4 knockdown ER-AsiSI U2OS cells were transfected with Flag-RIG-I, and then treated with 4-OHT to induce DSBs. Flag-RIG-I accumulation at DNA damage sites generated by AsiSI was detected by ChIP-qPCR. Data are presented as mean values +- SEM from three independent experiments. P values are determined by unpaired two-sided t -test. d Control and XRCC4 knockdown HEK293T cells overexpressing RIG-I were transfected with NHEJ reporter, and then cells were harvested for the NHEJ assay. Data are presented as mean values +- SEM from three independent experiments. P values are determined by unpaired two-sided t -test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 VAV2 is necessary in Ku70 and Ku80-mediated DNA NHEJ repair. a Fourteen top proteins potentially associate with VAV2 identified by mass spectrometry in ESCC cells ectopically overexpressing FLAG-tagged VAV2. Cell lysates were immunoprecipitated with antibody against FLAG and antibody against IgG was used as negative control. b Proteins potentially associate with VAV2 in KYSE30 and KYSE450 cells. Left panel is a Venn diagram showing 68 proteins identified in both cell lines. Right panel shows score of each protein. c - e Western blot detection of VAV2, Ku70 and Ku80 proteins by reciprocal immunoprecipitation with antibody against VAV2 ( c ), Ku70 ( d ) or Ku80 ( e ) in KYSE150 and KYSE450 cells. IgG was used as control. f Immunofluorescence analysis of VAV2 and Ku70 co-staining in KYSE150, KYSE450, PDC-4 and PDC-5 cells with or without IR (4 Gy), showing colocalization of VAV2 and Ku70. DAPI was used to label the nucleus. Scale bars, 20 um. g Multiple immunofluorescences analysis of VAV2, Ku70, and Ku80 in clinical ESCC tumor tissues. All tumors (T) show strong colocalization signal of the 3 proteins while the corresponding non-tumor tissues (N) from patient 3, which had low VAV2, show very weak colocalization signal. Merge 1 represents the merge of VAV2 and Ku70, Merge 2 represents the merge of VAV2 and Ku80 and Merge 3 represents the merge of VAV2, Ku70, and Ku80. Scale bars, 100 um. h Western blot analysis of FLAG-VAV2, Ku70 and Ku80 in ESCC cells ectopically express

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Protein-pulldown experiments point out that the E3 ubiquitin ligases RNF8 and RNF138 are required for radiation-related ubiquitination in G1 cells . ( a ) HeLa.S-Fucci cells proficient (wt) or deficient for RNF138 (RNF138 KO) and/or RNF8 (RNF8 KD) were irradiated either with 30 Gy X-rays or 30 Gy high-energy iron ions. 1 h after irradiation G1 cells were isolated via flow cytometry, their proteins extracted, and ubiquitinated proteins pulled down and visualized with TUBE2. PD: pull down; IB: immunoblot. ( b ) For quantification of ubiquitinated proteins, western-blot detection of Ku80 in the input-protein extracts (whole-cell extracts that were used for PD, see ( a ) was used for normalization to equal loading. For all conditions, the extracts for the pull-down reactions were performed from 10 6 cells each. Differences in the protein concentration of these extracts are based on the success of cell lysis and reflected in the Ku80 signal. n = 1.