Molecular interaction

(1) pGBKT7 Vector: Used for constructing Nuclear Yeast Two-Hybrid Bait-pGBKT7 plasmids or as an empty control.
(2) pGADT7 Vector: Used for constructing Nuclear Yeast Two-Hybrid and One-Hybrid Prey-pGADT7 plasmids or as an empty control.
(3) pABAi Vector: Used for constructing One-Hybrid Bait-pABAi plasmids.
(4) pGBKT7-53 Plasmid & pGADT7-T Plasmid: Used as a positive control pair for Nuclear Yeast Two-Hybrid verification and screening.
(5) pGBKT7-lam Plasmid & pGADT7-T Plasmid: Used as a negative control pair for Nuclear Yeast Two-Hybrid verification and screening.
(6) pOST1-NubI Plasmid & pBT3-N-bait Plasmid: Used as a functional validation control pair for Membrane Yeast Two-Hybrid verification and screening.
(7) pBT3-N Vector: Used for constructing bait proteins with the N-terminus in the cytosol and C-terminus in the organelle lumen (or extracellular space).
(8) pBT3-SUC Vector: Used for constructing bait proteins with the N-terminus in the organelle lumen (or extracellular space) and containing an N-terminal signal peptide.
(9) pBT3-STE Vector: Used for constructing bait proteins with the C-terminus in the cytosol, N-terminus in the organelle lumen (or extracellular space), and without an N-terminal signal peptide.
(10) pBT3-N and pBT3-STE: Both can be used for bait proteins with both N- and C-termini located in the cytosol.
(11) pPR3-N Vector: Used for constructing prey proteins with the C-terminus in the cytosol and N-terminus extracellular or in the organelle lumen.
(12) pPR3-C Vector: Used for membrane library construction and prey proteins without transmembrane domains.
(13) Ptsu2-APP Plasmid & pNubG-Fe65 Plasmid: Used as a positive control pair for Membrane Yeast Two-Hybrid verification and screening.
(14) Ptsu2-APP Plasmid & pPR3-N Plasmid: Used as a negative control pair for Membrane Yeast Two-Hybrid verification and screening.
(15) Y187 Strain: A GAL4 system yeast strain developed by Clontech for One-Hybrid and Two-Hybrid experiments. MATα type. Can be directly transformed with plasmids or used for library screening via mating with MATa strains (Y2HGold, AH109, etc.). Genotype: trp1, leu2. Reporter genes: lacZ, MEL1.
(16) Y1HGold Strain: A GAL4-AbA yeast One-Hybrid system strain developed by Clontech. MATα type. Can be directly transformed with plasmids for library screening. Genotype: ura3, leu2. Reporter gene: AbAr.
(17) Y2HGold Strain: A GAL4 system yeast Two-Hybrid strain developed by Clontech. MATa type. Can be directly transformed with plasmids or used for protein interaction verification/library screening via mating with MATα strains like Y187. Transformation markers: trp1, leu2. Reporter genes: AbAr, HIS3, ADE2, MEL1.
(18) NMY51 Strain: A yeast Two-Hybrid strain developed by DUALsystems BioTech for detecting membrane protein interactions. Can be directly transformed with plasmids for protein interaction verification or library screening. Genotype: *trp1, leu2-3*. Reporter genes: HIS3, ADE2, lacZ.

(1) The Yeast Two-Hybrid system is primarily used to study protein-protein interactions.

(2) The Yeast One-Hybrid system is primarily used to study DNA-protein interactions.

a. Promoter structure analysis: The promoter region sequence (typically ~2kb) is serially truncated or specific sites are mutated, then cloned into the luciferase reporter vector to detect promoter activity.
b. Promoter SNP analysis: Analyze the relative activity of single nucleotide polymorphisms (SNPs) present in the promoter region of some genes using the luciferase reporter system.
c. Verifying the interaction between a specific transcription factor and its regulatory sequence: Insert the sequence (usually the promoter region) into the reporter gene vector, and co-transfect an overexpression plasmid of the transcription factor into the experimental cells. Analyze whether transcription factor overexpression increases luciferase activity.
d. Analyzing signaling pathway activation: Clone the sequence of a downstream response element of the signaling pathway into the reporter gene vector. Under different upstream signal conditions, the luciferase activity represents the downstream response of the pathway. For example, in GPCR research, loading the cAMP response element (CRE) into a reporter gene vector and constructing a stable cell line can be used to analyze GPCR activation and inhibitor screening. Similarly, inserting the HIF1α response element, hypoxia-responsive element (HRE), into a luciferase reporter vector to create a stable cell line can be used for hypoxia-related pathway studies.
e. Verifying microRNA target sequences: Insert the candidate 3'UTR sequence into the reporter gene vector, then co-transfect the microRNA. A decrease in luciferase activity suggests it is a target sequence.

Firefly luciferase acts on luciferin in the presence of oxygen, magnesium, and ATP, while luciferase from the sea pansy (Renilla reniformis) acts on coelenterazine in the presence of oxygen. Dual-reporter assays combine Firefly luciferase and Renilla luciferase tests.

The composition of A, G, C, T differs, leading to different electrophoretic mobilities.The secondary structures of the DNA differ, leading to different electrophoretic mobilities.This phenomenon is more likely to occur with shorter Oligo DNAs; the differences between long-chain Oligo DNAs are smaller.

No,Ethidium bromide (EtBr) stains nucleic acids by intercalating between the bases in double-stranded helices. Synthetic DNA/RNA molecules are single-stranded. They can only be stained by EtBr if they form local hairpin loop structures through self-folding or form partial double-helix structures between strands. Because different products have different sequences, their ability to form double helices varies, and consequently, their staining capacity is not uniform. Therefore, the brightness of the EtBr-stained band cannot be used to quantify synthetic DNA/RNA.

1. Core Conclusion: A Co-Precipitation Band Alone Cannot Directly Prove Direct Interaction

    CoIP (Co-Immunoprecipitation) is a powerful technique for detecting protein-protein associations, but the presence of a co-precipitated candidate protein (observed as a Western blot band) only confirms that the two proteins are part of the same protein complex—it does not distinguish between "direct binding" (the two proteins interact with each other directly) and "indirect association" (the two proteins are linked via one or more intermediate proteins in the complex).​

2. Why Direct Interaction Cannot Be Confirmed by CoIP Alone​

    The working principle of CoIP determines this limitation:​

    CoIP relies on the specific binding of an antibody to the target protein to pull down (precipitate) the target protein from cell/tissue lysates.​

    During precipitation, the target protein “carries along” all other proteins that are physically associated with it—this includes not only proteins that bind directly to the target but also proteins that bind to other components of the target’s complex.​

For example:​

    If Protein A (target) binds to Protein B (intermediate), and Protein B binds to Protein C (candidate), CoIP using an anti-A antibody will pull down both B and C. The detected co-precipitation band of C only proves A and C are in the same complex, not that A and C bind directly.​

3. Key Follow-Up Experiments to Verify Direct Interaction​

    To confirm whether the target and candidate proteins interact directly, you must use techniques that eliminate intermediate proteins and test binding in a simplified system. Common complementary methods include:​

(1) GST Pull-Down Assay​

    Principle: Express the target protein as a fusion with GST (Glutathione S-Transferase) and immobilize it on glutathione beads. Incubate the beads with purified candidate protein (or a candidate protein fusion, e.g., His-tagged).​

    Logic: If the candidate protein binds to the GST-target fusion (but not to GST alone, a critical negative control), it confirms direct binding—since the system only contains the two purified proteins, no intermediates exist.​

(2) Far-Western Blotting​

    Principle: Separate the candidate protein by SDS-PAGE, transfer it to a membrane, and incubate the membrane with purified target protein (instead of a primary antibody). Detect the target protein with its specific antibody.​

    Logic: Direct binding between the target (in solution) and candidate (on the membrane) is required to produce a signal, ruling out indirect associations.​

(3) Fluorescence Resonance Energy Transfer (FRET) or Bioluminescence Resonance Energy Transfer (BRET)​

    Principle: Fuse the target and candidate proteins to two different fluorophores (FRET) or a luciferase and fluorophore (BRET). If the two proteins interact directly, the distance between the tags becomes <10 nm, triggering energy transfer (detectable as a fluorescence/bioluminescence signal shift).​

    Logic: Energy transfer only occurs when the two tags are in extremely close proximity, which is only possible if the proteins bind directly (not via intermediates).​

(4) Isothermal Titration Calorimetry (ITC) or Surface Plasmon Resonance (SPR)​

    Principle: These biophysical techniques measure the binding affinity and kinetics between two purified proteins in real time (e.g., ITC detects heat changes during binding; SPR measures refractive index shifts when one protein binds to another immobilized on a chip).​

    Logic: They quantify direct molecular interactions in a cell-free, intermediate-free system, providing definitive evidence of direct binding.

The probe design range can be determined by analyzing the promoter sequence. If the range is too broad, initially clone 0.5-1kb fragments into a luciferase reporter vector to verify promoter activity and whether it is regulated by the transcription factor. The core promoter can be identified through serial truncation validation. If the transcription factor under study has a known binding motif, the probe can be selected around that motif site. Alternatively, after performing ChIP or DNA pull-down to find enriched sequences, design segmented probes for validation.

1、Library Construction:
Total RNA extraction → mRNA isolation & purification → Double-stranded cDNA synthesis & purification → In vitro ligation of ds cDNA into pGADT7 vector → E. coli library culture → Library plasmids → Transformation into Y187 yeast host → Y187 yeast library culture.

2、Two-Hybrid Screening:
(1) Mating method:
Bait recombinant pGBKT7 plasmid construction → Autoactivation test: Co-transformation of bait recombinant plasmid & empty pGADT7 into Y2HGold yeast host → Mixing Y2HGold yeast culture containing bait with Y187 yeast library culture → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.
(2) Co-transformation method:
Bait recombinant pGBKT7 plasmid construction → Autoactivation test: Co-transformation of bait recombinant plasmid & empty pGADT7 into Y2HGold yeast host → Transformation of library plasmids into Y2HGold competent cells containing bait → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.

3、One-Hybrid Screening:
Bait recombinant pABAi plasmid construction → Autoactivation test: Transformation of linearized bait recombinant plasmid & empty pGADT7 into Y1HGold yeast host → Transformation of library plasmids into Y1HGold competent cells containing bait → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.

From the perspective of library construction:

(1) Flexible library construction methods: Depending on sample type, provided sample amount, etc., methods such as SMART, Gateway, In-Fusion in vitro recombination, and T4 DNA ligase in vitro ligation can be used for library construction.

(2) Controllable library insert size: The insert size can be controlled based on the type/size of protein the client wishes to study, to increase the possibility of screening for the target protein. For example, small molecular weight proteins like interleukins and growth factors; slightly larger molecular weight proteins like transcription factors and signaling pathway molecules.

(3) Libraries are universal for One- and Two-Hybrid screens: One type of library can be used for both One- and Two-Hybrid screening.

For library screening:

(1) Diverse screening methods: Library screening can be performed using mating or co-transformation.

(2) Controllable interaction strength: Screening pressure can be increased (or decreased) to screen for proteins with strong (or weak) interactions.

(3) Further validation of positive results: Positive results from library screening can undergo point-to-point verification to further exclude false positives.

The main service scope is as follows:

1、Nuclear Yeast One/Two-Hybrid library construction;

2、Membrane Yeast Two-Hybrid library construction;

3、Nuclear Yeast One/Two-Hybrid library screening;

4、Membrane Yeast Two-Hybrid library screening;

5、Nuclear Yeast One/Two-Hybrid point-to-point verification;

6、Membrane Yeast Two-Hybrid point-to-point verification

(1) HIS3. Y2HGold cannot synthesize histidine and therefore cannot grow on media lacking this essential amino acid. When bait and prey proteins interact, the expression of the Gal4-responsive His3 gene allows cells to biosynthesize histidine and grow on minimal medium. 3-AT can be added to suppress background.
(2) ADE2. Y2HGold also cannot grow on minimal medium lacking adenine. However, when two proteins interact, Ade2 expression is activated, enabling these cells to grow on Ade- minimal medium.
(3) MEL1. MEL1 codes for α-galactosidase, an enzyme naturally present in many yeasts. Due to the two-hybrid interaction, α-galactosidase (MEL1) is expressed and secreted by the yeast cells. Yeast colonies expressing Mel1 turn blue in the presence of the chromogenic substrate X-α-Gal.
(4) AUR1-C. a dominant mutant version of the AUR1 gene, encodes inositol phosphorylceramide synthase. *AUR1-C* is expressed in the Y2HGold yeast strain due to protein-protein interaction bringing the GAL4 transcription activation and DNA-binding domains into proximity. AbA can be added for background suppression.The pABAi vector carries the Ura3 gene. The pGADT7 vector carries the Leu gene. The pGBKT7 vector carries the Trp gene.

Plates used for screening:

Single Dropout: SD/-Ura, SD/-Leu /+AbA

Double Dropout: DDO [SD/-Leu/-Trp], DDO/X [SD/-Leu/-Trp/X-α-Gal]

Triple Dropout: TDO [SD/-Leu/-Trp/-His]

Quadruple Dropout: QDO [SD/-Leu/-Trp/-His/-Ade]

Luciferase sensitivity is 10-100 times higher than GFP, with a wider dynamic range, facilitating numerical analysis and comparison. It does not require a fluorescence microscope. In vivo, its luminescence has better tissue penetration than EGFP and other fluorescent proteins. Furthermore, due to the absence of endogenous activity, its background signal is very low.
The advantages of GFP and other fluorescent proteins compared to luciferase are the ability to perform subcellular localization and that observation does not require cell lysis, allowing for real-time observation.