Can the Next-Generation CRBN Binding Kit Usher in Another Spring for TPD?

Over the past decade, Targeted Protein Degradation (TPD) has evolved from a laboratory favorite to a 100-billion-yuan industry. Cereblon (CRBN), as one of the most commonly used E3 ligases, its ligand affinity data directly determines the activity window of PROTAC molecules. However, an overlooked technical detail—the labeling method of tracers—is quietly widening the gap between our results and the true objective values.

The high-throughput CRBN Binding Kit from the imported R brand pioneered the high-throughput screening paradigm using "thalidomide-fluorescent protein" as the tracer. This scheme has now been re-disassembled, verified, and upgraded by us. It is not a simple "domestic substitution" but a systematic repair of experimental distortion issues, enabling IC50 values to be closer to the objective true values for the first time.

Source of Distortion: When 105 kDa APC Protein Rides on 0.25 kDa Thalidomide

XL665, as the first-generation acceptor, is a modified allophycocyanin (APC) that couples APC subunits to enhance stability. It operates in the near-infrared region with an excitation wavelength of 620 nm and an emission wavelength of 665 nm, further reducing the interference of biological solutions on experiments (biological components rarely exhibit autofluorescence in the near-infrared region). When XL665 (molecular weight approximately 105 kDa) is used as an acceptor in CRBN Binding assays and conjugated with thalidomide, it exerts the following effects on the system:

Steric Hindrance

•         The diameter of APC/XL665 is approximately 6 nm, which is 10 times the length of thalidomide, forming a "fluorescent wall" at the entrance of the CRBN shallow pocket;

•         Macromolecular fluorescent labeling generates steric hindrance at the entrance of the CRBN shallow pocket, leading to a systematic overestimation of the apparent K_d by 1.5–4 fold.

•        

Diffusion Rate

•         The overall molecular weight of the protein tracer is high, resulting in a decrease in the diffusion coefficient D;

•         Small-molecule candidates (< 0.5 kDa) have a much higher diffusion coefficient than the labeled thalidomide in the kit, leading to an unbalanced competitive environment.

Result Distortion

•         The aforementioned factors cause deviations between IC50 values and actual results, affecting the selection of candidates.

Technological Breakthrough: Return Small Molecules to Small Molecules

Directly labeling thalidomide with small-molecule fluorophores with a molecular weight of less than 1 kDa physically eliminates the two major distortion factors. Competitive kinetics returns to a fair "small molecule vs. small molecule" track, ensuring that every IC50 value can withstand clinical scrutiny.

Let Data Speak

(1) Standard Curve

As shown in the standard curve chart of the Human CRBN Binding Assay (TR-FRET method, incubated at room temperature for 2 hours), the signal value decreases as the thalidomide standard competes with Thalidomide-Ac for binding to CRBN/DDB1. The coefficient of variation (CV) of each standard point is low, indicating high assay precision.

(2) Binding Activity and Inhibitor Verification*

Verification of the binding between Thalidomide-Ac and CRBN/DDB1 showed that when the protein concentration was constant, the detection signal increased with the increase of Thalidomide-Ac concentration. Lenalidomide, Pomalidomide, and PROTAC BET Degrader-1 could compete with Thalidomide-Ac for binding to CRBN/DDB1, confirming the specificity and reliability of the assay.

*Data for illustrative purposes only

Beyond CRBN: A One-Stop Solution for PROTAC

✦ TR-FRET-Based Ternary Complex Screening Kits

✦ PROTAC Target Proteins

✦ PROTAC Molecules – Positive Controls

✦ PROTAC Target-Related Antibodies

Brand Mission

ANT BIO PTE. LTD. is dedicated to advancing life science research by providing high-quality, reliable reagents and comprehensive solutions. We recognize the critical role of precise CRBN binding assays in accelerating TPD research and PROTAC development. Through our specialized sub-brands (Absin, Starter, UA), we have developed a full-spectrum product portfolio tailored to TPD research needs, covering CRBN binding kits, PROTAC target proteins, positive control molecules, and specific antibodies.

Our team adheres to stringent quality control standards throughout the product development and production process, ensuring the consistency, accuracy, and reliability of each product. We are committed to providing professional technical support and customer-centric services, helping researchers overcome experimental challenges such as assay distortion and accelerate the pace of TPD research breakthroughs. ANT BIO PTE. LTD. strives to be a trusted partner for scientists worldwide, contributing to the advancement of biomedicine through cutting-edge TPD research solutions.

Disclaimer

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ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.