Protein A/G magnetic beads feature a core-shell structure with a functional Protein A/G layer. This recombinant fusion protein is engineered to contain five highly homologous binding domains from Protein A and two from Protein G. This unique design confers significantly enhanced binding capacity for the IgG Fc region compared to standalone Protein A or G.

The beads broadly recognize IgG subclasses from human, mouse, and rat (e.g., human IgG1, IgG2, IgG3, IgG4; mouse IgG2a, IgG2b, IgG3; rat IgG2a, IgG2c). They also bind to total IgG from a wide range of other species, including cow, goat, sheep, rabbit, guinea pig, pig, dog, and cat.

Principle

Immunoprecipitation is a technique that exploits antigen-antibody specificity to isolate and enrich a target protein from a complex mixture for detection.

Experimental Procedure

Research Approach

1.Cell culture: KP-ICC and HuCCT-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1x penicillin/streptomycin (P/S) under conditions of 37°C and 5% CO2.

2.CRISPR screening and gene editing: Gene knockout and mutation were performed using CRISPR technology to screen for metabolism-related genes associated with Chemo-immunotherapy (CIT) resistance in the KP-ICC mouse model. It was found that Pck1 was significantly depleted in the CIT combination treatment group, suggesting its potential role in mediating treatment resistance.

3.The phosphorylation status of Pck1 determines its functional role: IP-MS revealed that Pck1 is phosphorylated by AKT at the S90 site. IP and WB experiments confirmed the phosphorylation of Pck1 at the S90 and T92 sites. It was found that pPck1 (phosphorylated at S90) promotes tumor cell tolerance to ferroptosis and diminishes the efficacy of CIT.

4.pPck1 regulates lactate metabolism by phosphorylating LDHA: IP-MS and co-localization experiments demonstrated that pPck1 interacts with LDHA and phosphorylates it at the T248 site. SPRING lactylation enhances the translocation of the SCAP-SREBP2 complex, activating the MVA pathway to synthesize antioxidants (CoQ10H2, MK4) and inhibit ferroptosis.

5.Targeting the pPck1-pLDHA-SPRINGlac axis enhances CIT efficacy: The use of a Pck1 phosphorylation-blocking peptide (PPB) or LDHA inhibitors to block this axis restored sensitivity to ferroptosis. Statins, which inhibit the MVA pathway, significantly enhanced the efficacy of CIT.

Sample

Cell lines: KP-ICC、HuCCT-1

Experimental Protocol of IP and Western blotting (WB) analysis

1.Cells were collected and lysed on ice using IP lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl, 1 mmol/L EDTA,1% Nonidet P-40 (NP-40), 10% glycerol).

2.Lysates were incubated with protein G agarose for 2 h and immunoprecipitated with corresponding antibodies overnight at 4℃.

3.The lysates were then incubated with protein A/G magnetic beads (HY-K0202) for 1 h, then washed 1 time using IP lysis buffer andwashed 3 times with IP washing buffer (300 mmol/L NaCl, 1.0 mmol/L EDTA, 25 mmol/L Tris-HCl, pH7.4, 1.0% NP-40).

4.The beads were eluted with 2x SDS-PAGE Sample Loading Buffer and subjected to WB analysis. The WB procedure was performed as previously described.

Experimental group setup

Conclusion

1.IP-MS identified S90 and T92 as phosphorylation sites on Pck1.

2.Validation confirmed that S90 is a key phosphorylation site of Pck1, and it was demonstrated to be the predominant phosphorylated form in K-ICC cells, indicating that the Pck1 kinase is in an active state in this cell line.

3.It was confirmed that the T248 site of LDHA is an important phosphorylation site, and phosphorylation at this site is critical for the enzymatic activity of LDHA.

Principle

If proteins A and B physically interact directly or reside within the same complex in vivo, IP of protein A using a specific antibody will co-precipitate protein B. This method enables the functional study of protein B through its known interactor, protein A.

Experimental Procedure

Research Approach

1.Bioinformatic Analysis: The TIDE algorithm was applied to analyze TCGA-LUAD data, revealing that high EHD1 expression is significantly associated with a higher risk of immune evasion. Kaplan-Meier analysis demonstrated that patients with high EHD1 expression had shorter overall survival, suggesting its potential as a poor prognostic factor.

2.EHD1 Regulation of T-cell Cytotoxic Activity: LUAD cell models with EHD1 knockdown (shEHD1) and overexpression (EHD1-OE) were established. Co-culture experiments with T cells were performed to assess the impact of EHD1 knockdown/overexpression on T-cell-mediated tumor cell killing.

3.Cell Culture: A549 and H1299 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, under standard culture conditions (37°C, 5% CO₂).

4.EHD1 Inhibits PD-L1 Degradation by Regulating Its Endocytic Recycling:Molecular docking and co-immunoprecipitation (Co-IP) confirmed a physical interaction between EHD1 and PD-L1.Immunofluorescence co-localization showed that EHD1 and PD-L1 co-localize in intracellular vesicles.Ubiquitination assays indicated that EHD1 suppresses K63-linked ubiquitination of PD-L1 (a signal for lysosomal degradation).Domain-mutation experiments identified the EH domain of EHD1 and the intracellular domain (ICD) of PD-L1 as critical regions for their interaction.Rescue experiments with mutants confirmed that only wild-type EHD1, but not the mutant lacking the EH domain, could restore PD-L1 expression and immune evasion function.

5.Upstream Regulatory Mechanism: YTHDF1 stabilizes EHD1 mRNA through m⁶A modification.

6.Validation in Animal Models: EHD1 knockdown enhances the efficacy of anti-PD-1 therapy.

Sample

Cell lines: A549、H1299

Experimental Protocol

Endogenous Co-IP : Conducting research on the naturally expressed proteins in cells or tissues.

1.Endogenous IP assays were performed by lysing untransfected cells with a specific lysis buffer.

2.The lysates were then centrifuged at 14,000 rpm for 15 min at 4℃ to eliminate cellular debris.

3.The supernatant was incubated with antibodies, including IgG, anti-EHD1 and anti-PD-L1 targeting the endogenous protein at 4℃.

4.Protein A/G beads were added, and the complex was pulled down and washed with phosphate-buffered saline with tween (PBST), then analyzed by WB.

Exogenous Co-IP: This method studies proteins expressed via a transfection-mediated exogenous expression system.

1.For exogenous IP assays, cells were transfected with plasmids encoding the overexpressed protein tagged with FLAG or Myc.

2.After incubation, cells were lysed, and the lysate was incubated with antibodies, including IgG, anti-Flag (20543-1-AP, Proteintech) and anti-Myc (16286-1-AP, Proteintech), specific to the tag at 4℃.

3.Protein A/G beads were added, and the complex was pulled down, washed with PBST, eluted, and analyzed byWestern blotting.

Experimental group setup

Conclusion

EHD1 directly interacts with PD-L1: It has been demonstrated that EHD1 binds to PD-L1 in lung cancer cell lines (A549 and H1299).

Immunoprecipitation (IP) is one of the primary applications of Protein A/G magnetic beads. It relies on the specific "lock-and-key" binding between an antigen and its antibody to "fish out" the target protein from complex samples! But if you want your experiment to go smoothly, you can't overlook these key points.

Sample Preparation

1.Adherent cells: Avoid enzymatic digestion methods. It is recommended to wash directly with PBS before proceeding with lysis to prevent interference from exogenous enzymes.

2.Suspension cells: After centrifugation, wash with PBS and proceed directly with lysis to minimize the introduction of external variables.

3.Tissue samples: Fresh samples are preferred. Avoid freezing or prolonged storage, as these may lead to dissociation of protein complexes.

Tips: Select an appropriate lysis buffer for sample processing. The lysis system should include protease inhibitors / phosphatase inhibitors. Maintain low-temperature conditions throughout the procedure to prevent protein degradation.

Pre-clearing

Incubate blank magnetic beads with the lysate for 30-60 min to remove non-specifically bound proteins and improve the signal-to-noise ratio.

Antibody Selection

1.Priority should be given to validated antibodies: Antibodies that have been "certified" in IP or ChIP experiments by the literature are significantly more reliable.

2.Antibody dosage: The antibody concentration should be tested in a gradient to determine the optimal amount, thereby avoiding high background caused by excessive use.

Experimental Group Setup