CRL4/CRBN Targeted Protein Degradation Complex Antibody Sampler Kit
cat.: HAK21095
Product Type: Antibody Sampler Kit
Species reactivity: Human
Applications: WB, IHC-P
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: PBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Uniprot #: SwissProt: Q96SW2 Human | Q8C7D2 Mouse | Q56AP7 Rat | Q13619 Human | Q16531 Human | Q3U1J4 Mouse | Q9ESW0 Rat | P62877 Human | P62878 Mouse | Q15843 Human | P29595 Mouse | Q71UE8 Rat | P0CG47 Human | P0CG48 Human | P62979 Human | P62987 Human | P0CG49 Mouse | P62991 Mouse | P0CG51 Rat | Q63429 Rat
Entrez Gene: 300084 Rat
Images
HAK21095_1.jpg Fig1: Western blot analysis of CRBN on different lysates with Rabbit anti-CRBN antibody (HA721457) at 1/1,000 dilution.

Lane 1: HEK-293 cell lysate
Lane 2: A375 cell lysate
Lane 3: A549 cell lysate
Lane 4: HeLa cell lysate
Lane 5: SH-SY5Y cell lysate
Lane 6: NIH/3T3 cell lysate
Lane 7: MCF7 cell lysate
Lane 8: Mouse brain tissue lysate
Lane 9: Rat testis tissue lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 51 kDa
Observed band size: 51 kDa

Exposure time: 40 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 (HA721457) 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.
HAK21095_2.jpg Fig2: Immunohistochemical analysis of paraffin-embedded human testis tissue with Rabbit anti-CRBN antibody (HA721457) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (HA721457) at 1/200 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.
HAK21095_3.jpg Fig3: CRBN was immunoprecipitated in 0.2mg HeLa cell lysate with HA721457 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA721457 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: HA721457 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of HA721457 in HeLa cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 1 minute 10 seconds; ECL: K1802
HAK21095_4.jpg Fig4: Western blot analysis of Cullin 4a on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET7106-56, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: Hela cell lysate
Lane 2: SiHa cell lysate
HAK21095_5.jpg Fig5: Immunocytochemistry analysis of HeLa cells labeling DDB1 with Rabbit anti-DDB1 antibody (ET1706-22) 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-DDB1 antibody (ET1706-22) 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.
HAK21095_6.jpg Fig6: Western blot analysis of DDB1 on different lysates with Rabbit anti-DDB1 antibody (ET1706-22) at 1/2,000 dilution.

Lane 1: MCF7 cell lysate
Lane 2: HeLa cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: Neuro-2a cell lysate
Lane 5: PC-12 cell lysate
Lane 6: C6 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 127 kDa
Observed band size: 127 kDa

Exposure time: 2 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 (ET1706-22) 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.
HAK21095_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-DDB1 antibody (ET1706-22) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (ET1706-22) 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.
HAK21095_8.jpg Fig8: Western blot analysis of RBX1 on different lysates with Rabbit anti-RBX1 antibody (HA722779) at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: Jurkat cell lysate (20 µg/Lane)
Lane 3: HEK-293 cell lysate (20 µg/Lane)
Lane 4: HepG2 cell lysate (20 µg/Lane)
Lane 5: MCF7 cell lysate (20 µg/Lane)
Lane 6: NIH/3T3 cell lysate (20 µg/Lane)
Lane 7: RAW264.7 cell lysate (20 µg/Lane)
Lane 8: PC-12 cell lysate (20 µg/Lane)
Lane 9: C6 cell lysate (20 µg/Lane)
Lane 10: Mouse heart tissue lysate (30 µg/Lane)
Lane 11: Rat heart tissue lysate (30 µg/Lane)

Predicted band size: 12 kDa
Observed band size: 16 kDa

Exposure time: 14 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 (HA722779) 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.
HAK21095_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded human breast tissue with Rabbit anti-RBX1 antibody (HA722779) 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 (HA722779) 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.
HAK21095_10.jpg Fig10: Immunocytochemistry analysis of Jurkat cells labeling RBX1 with Rabbit anti-RBX1 antibody (HA722779) at 1/1,000 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-RBX1 antibody (HA722779) at 1/1,000 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.
HAK21095_11.jpg Fig11: RBX1 was immunoprecipitated from 0.2 mg HeLa cell lysate with HA722779 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA722779 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: HA722779 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of HA722779 in HeLa cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 2 minutes 22 seconds; ECL: K1801
HAK21095_12.jpg Fig12: Western blot analysis of NEDD8 on different lysates with Rabbit anti-NEDD8 antibody (ET1702-84) at 1/1,000 dilution.

Lane 1: COS-1 cell lysate
Lane 2: HeLa cell lysate
Lane 3: Raji cell lysate
Lane 4: HepG2 cell lysate
Lane 5: K-562 cell lysate
Lane 6: RAW264.7 cell lysate
Lane 7: NIH/3T3 cell lysate
Lane 8: PC-12 cell lysate
Lane 9: C6 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 9 kDa
Observed band size: 9-90 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 (ET1702-84) 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.
HAK21095_13.jpg Fig13: Immunohistochemical analysis of paraffin-embedded rat colon tissue with Rabbit anti-NEDD8 antibody (ET1702-84) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (ET1702-84) 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.
HAK21095_14.jpg Fig14: Western blot analysis of Ubiquitin on different lysates with Rabbit anti-Ubiquitin antibody (ET1609-21) at 1/5,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 10μM MG-132 for 6 hours cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: NIH/3T3 treated with 10μM MG-132 for 8 hours cell lysate
Lane 5: PC-12 cell lysate

Lysates/proteins at 20 µg/Lane.

Exposure time: 1 minute 34 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 (ET1609-21) at 1/5,000 dilution and competitor's antibody at 1/2,000 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.
HAK21095_15.jpg Fig15: Immunocytochemistry analysis of HepG2 cells labeling Ubiquitin with Rabbit anti-Ubiquitin antibody (ET1609-21) at 1/500 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-Ubiquitin antibody (ET1609-21) 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.
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