CRL4/CRBN Targeted Protein Degradation Complex Antibody Sampler Kit
Catalog# HAK21095
CRL4/CRBN Targeted Protein Degradation Complex Antibody Sampler Kit
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WB
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IHC-P
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Human
Overview
Kit Components
| Product Includes | Specification | Application | Reactivity | Mw |
|---|---|---|---|---|
| CRBN[HA721457] | 20µl | WB,IHC-P,IP | Human,Mouse,Rat | Predicted band size: 51 kDa |
| Cullin 4a[ET7106-56] | 20µl | WB,IHC-P | Human | Predicted band size: 88 kDa |
| DDB1[ET1706-22] | 20µl | WB,IF-Cell,IF-Tissue,IHC-P,IP,FC | Human,Mouse,Rat | Predicted band size: 127 kDa |
| RBX1[HA722779] | 20µl | WB,IHC-P,IF-Cell,IP,IF-Tissue | Human,Mouse,Rat | Predicted band size: 12 kDa |
| NEDD8[ET1702-84] | 20µl | WB,IF-Cell,IF-Tissue,IHC-P,IP | Human,Mouse,Rat,Monkey | Predicted band size: 9 kDa |
| Ubiquitin[ET1609-21] | 20µl | WB,IF-Cell,IF-Tissue,IHC-P,FC | Human,Mouse,Rat | |
| Goat Anti-Rabbit IgG (H+L)[HA1001] | 100µl | WB,ELISA,IHC-P | Rabbit |
Product Description
The CRL4/CRBN Targeted Protein Degradation Complex Antibody Sampler Kit provides an economical means of detecting the individual components of a CRL4/CRBN E3 ubiquitin ligase complex, including free and conjugated forms of both NEDD8 and ubiquitin.
Product Features
Storage Buffer
PBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Storage Instructions
Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Background
Targeted protein degradation is an experimental method of drug-based protein targeting that leverages endogenous proteolysis machinery, such as the the ubiquitin proteasome system (UPS), to selectively degrade proteins of interest (POIs). It is being actively explored as a therapeutic strategy to target and degrade POIs that contribute to disease progression. This approach differs from traditional small-molecule therapeutics that seek to suppress disease proteins (e.g., kinases) by sterically blocking catalytic domains. Proteolysis-targeting chimeras (PROTACs) and "molecular glue" degraders are the two primary degrader modalities used in UPS-mediated targeted protein degradation. PROTACs are bivalent, chemically-linked ligands that induce proximity between a POI and an E3 ubiquitin ligase, resulting in ubiquitination of the target protein, and its subsequent degradation by the UPS. Molecular glues are molecules that chemically generate novel interaction surfaces between two proteins, which can used to induce proximity between a POI and an E3 ligase. Cereblon (CRBN) is the substrate-recognition component of a Cullin-RING-ubiquitin (E3) ligase complex (CRL4/CRBN) that was among the first to be recognized for its therapeutic potential via targeted protein degradation. The CRL4/CRBN complex is comprised of CRBN, DDB-1, RBX1, and the scaffold protein CUL4A; its ligase activity is dynamically regulated via the covalent modification (neddylation) of CUL4A by NEDD8. In unrelated mechanistic studies of multiple myeloma drugs, it was revealed that phthalimides (e.g., thalidomide, lenalidomide) promoted CRBN-dependent recruitment, ubiquitination, and proteasomal degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3).
Data Links
Background References
1. Bond, M.J. and Crews, C.M. (2021) RSC Chem Biol 2, 725-742.
2. Sakamoto, K.M. et al. (2001) Proc Natl Acad Sci U S A 98, 8554-9.
3. Sakamoto, K.M. et al. (2003) Mol Cell Proteomics 2, 1350-8.
4. Krönke, J. et al. (2014) Science 343, 301-5.
5. Hofmann, H. et al. (2013) J Virol 87, 11741-50.
6. Lu, G. et al. (2014) Science 343, 305-9.
7. Shirasaki, R. et al. (2021) Cell Rep 34, 108532.
8. Alabi, S.B. and Crews, C.M. (2021) J Biol Chem 296, 100647.
9. Alabi, S. et al. (2021) Nat Commun 12, 920.
Images
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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. -
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) (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 (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. -
CRBN was immunoprecipitated in 0.2mg HeLa cell lysate with HA721457 at 2 µg/10 µl beads. 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 -
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 -
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. -
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. -
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) (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 (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. -
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. -
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. -
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. -
RBX1 was immunoprecipitated from 0.2 mg HeLa cell lysate with HA722779 at 2 µg/10 µl beads. 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 -
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. -
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) (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 (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. -
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. -
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.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
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