NF-κB Family Antibody Sampler Kit
cat.: HAK21141
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.
Uniprot #: SwissProt: Q04206 Human | Q04207 Mouse | Q04206 Human | Q04207 Mouse | P19838 Human | P25799 Mouse | Q63369 Rat | Q01201 Human | Q04864 Human | Q00653 Human | Q9WTK5 Mouse
Entrez Gene: 309165 Rat | 309452 Rat
Images
HAK21141_1.jpg Fig1: Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/5,000 dilution.

Lane 1: A549 cell lysate
Lane 2: MCF7 cell lysate
Lane 3: HeLa cell lysate
Lane 4: RAW264.7 cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 65 kDa
Observed band size: 65 kDa

Exposure time: 49 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 (ET1603-12) at 1/5,000 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.
HAK21141_2.jpg Fig2: Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/5,000 dilution.

Lane 1: HAP1-parental cell lysate
Lane 2: HAP1-NF-kB p65 KD cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 65 kDa
Observed band size: 65 kDa

Exposure time: 60 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 (ET1603-12) at 1/5,000 dilution was used in 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.
HAK21141_3.jpg Fig3: Western blot analysis of Phospho-NF-kB p65 (S536) on different lysates with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (HA723223) at 1/5,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HeLa treated with 100nM Calyculin A and 20ng/mL TNF-α for 10 minutes cell lysate (20 µg/Lane)
Lane 3: NIH/3T3 cell lysate (20 µg/Lane)
Lane 4: NIH/3T3 treated with 100nM Calyculin A and 20ng/mL TNF-α for 10 minutes cell lysate (20 µg/Lane)

Predicted band size: 60 kDa
Observed band size: 65 kDa

Exposure time: 59 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 (HA723223) 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.
HAK21141_4.jpg Fig4: Western blot analysis of NF-kB p105 / p50 on different lysates with Rabbit anti-NF-kB p105 / p50 antibody (ET1603-18) at 1/5,000 dilution.

Lane 1: HCT 116-si NT cell lysate
Lane 2: HCT 116-si NFKB1 cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 105/50 kDa
Observed band size: 105/50 kDa

Exposure time: 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 (ET1603-18) 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.
HAK21141_5.jpg Fig5: Western blot analysis of NF-kB p105 / p50 on different lysates with Rabbit anti-NF-kB p105 / p50 antibody (ET1603-18) at 1/5,000 dilution.

Lane 1: THP-1 cell lysate
Lane 2: Raji cell lysate
Lane 3: Daudi cell lysate
Lane 4: MCF7 cell lysate
Lane 5: NIH/3T3 cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 105/50 kDa
Observed band size: 105/50 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 (ET1603-18) 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.
HAK21141_6.jpg Fig6: Western blot analysis of c-Rel on different lysates with Rabbit anti-c-Rel antibody (ET1705-44) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: K-562 cell lysate
Lane 3: Raji cell lysate
Lane 4: Daudi cell lysate
Lane 5: Jurkat cell lysate
Lane 6: A431 cell lysate
Lane 7: U-937 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 69 kDa
Observed band size: 75 kDa

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 (ET1705-44) 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.
HAK21141_7.jpg Fig7: Western blot analysis of NF-kB p100 / NFKB2 on different lysates with Rabbit anti-NF-kB p100 / NFKB2 antibody (ET1705-45) at 1/1,000 dilution.

Lane 1: MCF7 cell lysate (20 µg/Lane)
Lane 2: THP-1 cell lysate (20 µg/Lane)
Lane 3: Mouse kidney tissue lysate (30 µg/Lane)
Lane 4: Rat heart tissue lysate (30 µg/Lane)
Lane 5: Rat spleen tissue lysate (30 µg/Lane)

Predicted band size: 97 kDa
Observed band size: 110 kDa

Exposure time: 20 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 (ET1705-45) 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.
HAK21141_8.jpg Fig8: Immunocytochemistry analysis of HeLa cells treated with or without 50 ng/mL TNF-alpha for 20 minutes labeling NF-kB p65 with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/400 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/400 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. 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.
HAK21141_9.jpg Fig9: Immunocytochemistry analysis of HeLa cells untreated / treated with 100nM Calyculin A and 20ng/mL TNF-α for 15 minutes labeling Phospho-NF-kB p65 (S536) with Rabbit anti-Phospho-NF-kB p65 (S536) antibody (HA723223) 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-Phospho-NF-kB p65 (S536) antibody (HA723223) 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.
HAK21141_10.jpg Fig10: Immunocytochemistry analysis of NIH/3T3 cells labeling NF-kB p105 / p50 with Rabbit anti-NF-kB p105 / p50 antibody (ET1603-18) 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-NF-kB p105 / p50 antibody (ET1603-18) 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.
HAK21141_11.jpg Fig11: Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue with Rabbit anti-NF-kB p105 / p50 antibody (ET1603-18) 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 (ET1603-18) 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.
HAK21141_12.jpg Fig12: Flow cytometric analysis of NIH/3T3 cells labeling NF-kB p105 / p50.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1603-18, 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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