MBP and MBP+hBT proteins were immunoprecipitated from bacterial lysates and mouse serum samples by Amylose resin, MBP antibody (New England BioLabs), or human Betatrophin antibody (Phoenix Pharmaceuticals). Two μg of protein were run on 4–15% SDS-PAGE gel and transferred to nitrocellulose membrane. Coomassie blue staining was used to identify MBP (42 kDa) and MBP+hBT (64 kDa).
Mbp antibody
Manufactured by New England Biolabs
The MBP antibody is a laboratory tool used for the detection and identification of maltose-binding protein (MBP) in various biological samples. It is a highly specific and sensitive antibody that binds to the MBP protein, enabling researchers to analyze and quantify its presence in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Amylose resin, by New England Biolabs (1 mentions)
Amylose resin, by Merck Group (1 mentions)
Anti 6his, by Merck Group (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Enduro gds imaging system, by Corning (1 mentions)
Cm5 chip, by GE Healthcare (1 mentions)
Biacore t200, by GE Healthcare (1 mentions)
Biaevaluation software, by GE Healthcare (1 mentions)
Glutathione (gsh), by GE Healthcare (1 mentions)
6 protocols using mbp antibody
1
Immunoprecipitation and SDS-PAGE Analysis
2
Protein Interaction Mapping by Blot Overlay
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Protein interactions were assessed in vitro by blot overlay assays, as previously described [16 (link),53 (link),57 (link),60 (link),62 (link)]. Briefly, purified GST, GST-Hsp20 and phosphorylated GST-Hsp20 recombinant proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Following blocking, the membranes were incubated with MBP-14-3-3 fusion protein. The blots were probed with anti-MBP and the immunoreactive bands were visualized using ECL reagents. In another set of experiments, GST, GST-CFL2 and phosphorylated GST-CFL2 recombinant proteins were allowed to interact with MBP-14-3-3, as described above.
In a different set of experiments, mapping of the minimal binding domain of 14-3-3 to CFL2 was performed using equal amounts of GST, GST-CFL2 (amino acids 1–166), GST-CFL2 (amino acids 1–55), GST-CFL2 (amino acids 19–154) and GST-CFL2 (amino acids 105–166) proteins that were separated by SDS-PAGE and allowed to interact with MBP-14-3-3 (amino acids 1–247), as described above. Western blot analysis with an MBP antibody (New England Biolabs) determined the minimal region of CFL2 required for 14-3-3 binding.
In parallel, GST, GST-CFL2 and phosphorylated GST-Hsp20 recombinant proteins were allowed to interact with MBP-14-3-3 (amino acids 1–120) or MBP-14-3-3 (amino acids 115–247), as described above.
In a different set of experiments, mapping of the minimal binding domain of 14-3-3 to CFL2 was performed using equal amounts of GST, GST-CFL2 (amino acids 1–166), GST-CFL2 (amino acids 1–55), GST-CFL2 (amino acids 19–154) and GST-CFL2 (amino acids 105–166) proteins that were separated by SDS-PAGE and allowed to interact with MBP-14-3-3 (amino acids 1–247), as described above. Western blot analysis with an MBP antibody (New England Biolabs) determined the minimal region of CFL2 required for 14-3-3 binding.
In parallel, GST, GST-CFL2 and phosphorylated GST-Hsp20 recombinant proteins were allowed to interact with MBP-14-3-3 (amino acids 1–120) or MBP-14-3-3 (amino acids 115–247), as described above.
3
BASL-YDA Protein Interaction Analysis
4
Crosslinking of GR-loading Complex
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To ensure that Hop crosslinks the bound segment in the context of the loading complex, crosslinking reactions were performed immediately after the complex was fractionated from SEC (see the above complex preparation section). Using a UV-transparent, 96well microplate (Corning) as a fraction collector, the whole fractions of the eluted GRloading complex were subjected to UV exposure using an agarose gel imaging system (Enduro GDS Imaging System). Samples were irradiated for 60 mins in total. To prevent overheating, the 96-well plate was placed on a shallow plate filled with constantly refreshed ice water during the time course of the exposure. SDS-PAGE was used to analyze cross-linked product, followed by Western plot transfer to nitrocellulose and probed with an MBP antibody (New England BioLabs) (Extended Data Fig. 27).
5
Optical Biosensor Analysis of Calcium-Dependent Protein Interactions
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Experiments were performed with a Biacore T200 optical biosensor instrument with CM5 chips (GE Healthcare, Biacore) at 28 or 5°C as indicated (Wang et al, 2016 (link)). All the proteins used in SPR were buffer‐exchanged to HBS‐P buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% surfactant P20) with 5 mM CaCl2 or 10 mM EGTA, which was filtered through 0.22 μM micro membrane. MBP‐OsCRT3 or MBP‐OsCBL8 was captured by the immobilized MBP antibodies (New England Biolabs, E8038) on a CM5 sensor surface using standard amine‐coupling procedures. MBP antibody in coupling buffer (10 mM sodium acetate, pH 5.0) was injected on the surface and remaining activated groups were blocked with 1 M ethanolamine, pH 8.5. MBP protein was captured in the control channel, which was used as a reference surface. The GST‐OsCIPK7‐C protein was serially diluted by HBS‐P buffer to a serial of concentrations as indicated. The proteins were then flowed over the chip surface and the response units were measured. The true binding response was obtained by subtracting the value of control channel to eliminate the bulk effects. Results were interpreted using the BiaEvaluation software using 1:1 binding model (GE Healthcare).
6
Purification and Interaction Analysis of GST-NHSL1 and Abi Proteins
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GST and MBP fusion proteins were purified from BL21-CodonPlus (DE3)-RP E.
coli (Stratagene) using glutathione (GE Healthcare) or amylose (New England Biolabs, Inc.) beads. Purified GST-NHSL1 fragments were separated on SDS-PAGE and transferred to PVDF membranes and overlayed as described previously 40 with purified MBP-Abi full-length or MBP-Abi-delta-SH3, and MBP was detected with MBP antibodies (New England Biolabs).
coli (Stratagene) using glutathione (GE Healthcare) or amylose (New England Biolabs, Inc.) beads. Purified GST-NHSL1 fragments were separated on SDS-PAGE and transferred to PVDF membranes and overlayed as described previously 40 with purified MBP-Abi full-length or MBP-Abi-delta-SH3, and MBP was detected with MBP antibodies (New England Biolabs).
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