Trimeric wild-type HA or COBRA HA ectodomains were expressed and purified in Expi293F cells following the manufacturer’s protocol and as previously described (26 ). Collected supernatants containing the HA antigens were purified on a HisTrap Excel column following the manufacturer’s recommended protocol. Eluted fractions were pooled and purified proteins were verified for integrity by probing with an anti-HIS tag antibody (Biolegend) as well as with subtype-specific mAbs via SDS-PAGE and Western blot.
Anti his tag antibody
Manufactured by BioLegend
Sourced in United States
The Anti-His tag antibody is a laboratory reagent that specifically recognizes and binds to the histidine tag (His-tag) sequence, which is commonly used to facilitate the purification and detection of recombinant proteins. The antibody can be used in various immunological techniques, such as Western blotting, immunoprecipitation, and ELISA, to identify and monitor the presence of His-tagged proteins.
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Lab products found in correlation
5 protocols using anti his tag antibody
1
Recombinant HA Antigen Production
2
COBRA2 HA Antigen Protein Production
3
Enumeration of Antigen-Specific ASC
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For enumeration of antigen-specific ASC, 6XHis-tagged protein antigens were coated directly into 70% (v/v) EtOH pre-conditioned assay wells at 10 μg/mL in PBS overnight at 4 °C. Alternatively, 6XHis-tagged protein solutions at 10 µg/mL in PBS (unless otherwise specified) were applied to EtOH pre-conditioned wells precoated overnight at 4 °C with 10 µg/mL (unless otherwise specified) anti-His tag antibody (Biolegend, San Diego, CA, USA) and incubated overnight at 4 °C to improve antigen absorption. Following one wash with 150 μL PBS, plates were blocked with 150 μL complete BCM for 1 h at room temperature prior to addition of polyclonally-stimulated PBMC (1–3 × 105 cells/well). Plates were then incubated for 16 h at 37 °C, 5% CO2, and SFU were visualized using the human IgA/IgG/IgM Three-Color ImmunoSpot® kit (from CTL) according to the manufacturer’s instructions.
4
Reticulocyte Binding Assay with MSP1P-19
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The reticulocytes were prepared as described by Gruszczyk et al. (14 (link)). Briefly, enriched reticulocytes resuspended in PBS in the presence or absence of antibodies were incubated with recombinant MSP1P-19 proteins for 1 h at RT. After being washed for three times, the binding complexes were incubated with anti-His tag antibody (BioLegend) for 20 min on ice, shielded from light. After washing, thiazole orange (Solarbio) was added and incubated for 20 min. The reticulocytes were then collected and resuspended in PBS for further analysis on Accuri C6 plus flow cytometer (BD Biosciences). One hundred thousand events were collected and analyzed by FlowJo software.
5
Flow Cytometry Analysis of Dual CAR T Cells
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All samples were analyzed on a BD LSR II (BD, Franklin Lakes, NJ, USA) or CytoFLEX (Beckman Coulter, Inc, Brea, CA, USA) flow cytometer using BD FACSDiva software (BD). For ALK surface expression, the median fluorescence intensity was calculated and compared with controls. In all cases, OneComp eBeads compensation beads (Thermo Fisher Scientific) were stained with the relevant antibodies as per the manufacturer's instructions and used for instrument compensation to prevent fluorescence spillover. All results were analyzed using FlowJo v10 software (BD). TE9 in ALK/B7H3 dual CARs was detected by primary incubation with recombinant human B7-H3 (4Ig)/B7-H3b protein with a C-terminal 10£ His tag (R&D Systems Inc, Minneapolis, MN, USA) and secondary staining with anti-His tag antibody (BioLegend, San Diego, CA, USA). Other antibodies used in flow cytometry are listed in supplementary Table 3.
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