The polyclonal antibodies against Mora and the MBP were produced using MBP and MBP-Mora as immunogens. MBP–Mora and MBP were produced in E. coli using the pMAL Protein Fusion and Purification System (New England BioLabs) and affinity purified on amylose resin columns. Rabbits were immunised by repeated intramuscular injections of the purified fusion proteins (500 μg) suspended in Complete Freund Adjuvant. The specificity of the antiserum against Mora was tested by western blotting on protein extracts from wild-type and mora mutant flies. The specificity of the antiserum against MBP was tested by western blotting on protein extracts from E. coli expressing or not expressing MBP. The anti-Mora antibodies were affinity-purified from rabbit serum using a Sepharose column coupled to glutathione S-transferase (GST)–Mora produced in E.coli using the GST Gene Fusion System (GE Healthcare Life Sciences) and eluted with glycine-HCl (pH 3). The anti-MBP antibodies were purified using the same protocol on a Sepharose column coupled to MBP.
Pmal protein fusion and purification system
Manufactured by New England Biolabs
Sourced in United States
The PMAL Protein Fusion and Purification System is a laboratory tool designed to facilitate the expression and purification of recombinant proteins. It enables the attachment of a Maltose Binding Protein (MBP) tag to target proteins, which can then be efficiently purified using affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Pmal c5x, by New England Biolabs (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Amylose resin, by New England Biolabs (2 mentions)
Snakeskin dialysis tubing, by Thermo Fisher Scientific (2 mentions)
Pmal c2x, by New England Biolabs (2 mentions)
Gst gene fusion system, by GE Healthcare (1 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti trim56 pab, by Fortis Life Sciences (1 mentions)
Rabbit anti mbp pab, by New England Biolabs (1 mentions)
Pmal c5x vector, by New England Biolabs (1 mentions)
Kta fplc system, by Cytiva (1 mentions)
Mbptrap hp column, by Cytiva (1 mentions)
Complete freund s adjuvant, by BD (1 mentions)
Z fr amc, by Bachem (1 mentions)
Freund s adjuvant, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Chemiluminescent emsa kit, by Beyotime (1 mentions)
Maxi plasmid isolation kit, by Qiagen (1 mentions)
Amylose agarose beads, by New England Biolabs (1 mentions)
23 protocols using pmal protein fusion and purification system
1
Polyclonal Antibody Production and Purification
2
Proteomic Analysis of M. agalactiae
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M. agalactiae grown under normal and mating growth conditions was subjected to proteomic analyses. Cells were collected by centrifugation of mycoplasma cultures (8,000 × g), washed, and resuspended in Dulbecco’s phosphate-buffered saline (DPBS). Proteins were separated by one-dimensional (1D) SDS-PAGE, and gel sections were subjected to trypsin digestion. Peptides were further analyzed by nano liquid chromatography coupled to a nanospray Q-Exactive hybrid quadruplole-Orbitrap mass spectrometer (Thermo Scientific). Peptides were identified as previously described by using a database consisting of M. agalactiae strain 5632 entries (26 (link)). ICEA products were detected by specific antiserums on Western and colony blots (25 (link), 27 (link)). Triton-X114-soluble proteins were extracted from M. agalactiae as previously described (28 (link)). The anti-CDS14 lipoprotein rabbit serum was produced by animal immunization with a recombinant CDS14 protein (pMAL protein fusion and purification system; New England Biolabs). A sheep serum raised against the M. agalactiae P80 surface antigen was used as a control (25 (link)). Western and colony blotting was developed by using swine anti-rabbit or rabbit anti-sheep immunoglobulin G conjugated to horseradish peroxidase (DAKO) and 4-chloro-naphthol substrate or SuperSignal West Dura extended-duration substrate (Thermo Scientific).
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3
Characterizing Protein-Protein Interactions
4
Affinity Purification of Polyclonal Antibodies
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Polyclonal antibodies against the carboxy termini of classical and SOL calpain were raised in rabbit using the following epitopes: classical: GFAPYDIDSFIQATMYS, SOL: ISPQMESIHQPRPYV. To affinity purify the antibodies, the pMAL Protein Fusion and Purification System (New England Biolabs) was used to express a fusion protein consisting of the epitope peptide fused to maltose-binding protein in Escherichia coli. The fusion protein was then purified by incubation with amylose beads, subjected to SDS-PAGE and transferred to a PVDF membrane, which was then incubated with the antibody-containing serum overnight at 4°C. After three washes with 50 mM Tris pH 7.4 with 500 mM NaCl and one wash with 50 mM Tris pH 7.4 with 100 mM NaCl, bound antibody was eluted from the membrane with Glycine 50 mM pH 2.5. After adjusting to pH 7 or 8 with Tris 2 M pH 8.0, antibodies were concentrated, glycerol added to a final concentration of 50% and aliquots frozen at −80°C.
5
TRIM56 Interaction with ZIKV RNA
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To assess whether TRIM56 directly interacts with ZIKV RNA, we first purified MBP-tagged T56-C392 (comprising the C-terminal 392 aa of human TRIM56) protein from E. coli using the pMAL Protein Fusion and Purification System (New England Biolabs, E8000S, USA). To produce a control protein, we purified MBP along with a short stretch of the downstream polylinker (MBP-polylinker) from E. coli transformed with the empty vector pMAL-c4x. ZIKV RNA was extracted using TRIzol from virions in high-titer ZIKV stocks. Subsequently, two micrograms of MBP-polylinker or MBP-T56-C392 protein were incubated with 0.5 μg of ZIKV RNA at room temperature for 30 min, followed by pull-down of MBP-tagged proteins using amylose resin. After three washes, the resins were divided equally into two fractions, which were subjected to RNA isolation by TRIzol and protein extraction by boiling in SDS sample buffer, respectively. Quantification of ZIKV RNA levels was performed by qPCR as described above. MBP-polylinker and MBP-T56-C392 in the protein samples were probed by immunoblotting using rabbit anti-MBP pAb (New England Biolabs) and rabbit anti-TRIM56 pAb (Bethyl Labs).
6
Antibody Generation for Visual Proteins
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The antibodies against the lamprey parietopsin and parapinopsin were generated against the C-terminal 55-, 50-amino-acid region of the proteins, respectively. The anti-β-arrestin antibody was generated against 269 amino acids (V29–E297) of the protein. The antigens were prepared using the pMAL Protein Fusion and Purification System (New England Biolabs), following the previously reported methods [10 (link)], and immunized to the chicken (anti-parietopsin antibody), mouse (anti-parapinopsin antibody), and guinea pig (anti-β-arrestin antibody). Antiserums obtained from these immunized animals were used for immunohistochemistry. The anti-VGLUT (Synaptic Systems, #135 503), anti-Go-type G protein (MBL, #551), and anti-Gt/gust-type G protein (CytoSignal, #TF15) antibodies were commercially obtained. Antibodies against iguana parapinopsin and parietopsin were previously generated [10 (link)].
7
Purification and Ubiquitination Assay of VyRCHC114 E3 Ligase
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The open reading frame (ORF) of VyRCHC114 and the different site mutants C320S, H341A, C328S, and N355A were separately cloned into the SalI/KpnI site of the pMAL-c5X vector (New England Biolabs UK Ltd, Hitchin, UK). According to the manufacturer’s instructions, the pMAL protein fusion and purification system (New England Biolabs) was used to purify the fusion protein. Ubiquitination activity was then measured that according to the method described above [66 (link)], albeit with the following modifications made: 250 ng of purified E3 (MBP-VyRCHC114, C320S, H341A, C328S, and N355A) in the ubiquitination buffer (50 mM Tris-HCl (pH 7.5), while the other reagents and steps used were the same. Primer sequence information in Table S1 .
8
Purification and Characterization of HsfB2b DNA Binding Domain
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Coding sequence encoding DNA binding domain of HsfB2b was fused to pMAL-c2 vector. The MBP and MBP-HsfB2bDBD proteins were expressed in Escherichia coli BL21 strain according to the manufacturer’s instructions using the pMAL Protein Fusion and Purification System (#E8200; New England BioLabs) and purified using MBPtrap HP column (Cytiva) attached to ÄKTA FPLC system (Cytiva). The Cy5-labeled probes (HSE, 5′-Cy5- TTTCCTCCTTAG AA ACAT C G AA AAAACAAAAGGAGAGA-3′; mHSE, 5′-Cy5- TTTCCTCCTTAA AA ACA TT TAA AA AAAACAAAAGGAGAGA -3′) and unlabeled competitors were generated by annealing 40 bp-length oligonucleotides. 5 μM of purified proteins and 100 nM of Cy5-labeled probe were incubated at room temperature in binding buffer (10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 5% glycerol and 5 mM DTT). For competition assay, 100-fold molar excess of each competitor was added to the reaction mixture before incubation. The reaction mixtures were resolved by electrophoresis through 6% polyacrylamide gel in 0.5X Tris–borate EDTA buffer at 100 V. The Cy5 signals were detected using WSE-6200H LuminoGraph II (ATTO).
9
Generation of Monoclonal Antibodies against Active β7
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DNA encoding N-terminal 1–458 amino acids of murine β7 was subcloned into a pMALc2x vector and that vector was transformed into E.coli BL21 competent cells. To synthesize recombinant maltose binding protein (MBP)-β7, BL21 were cultured at 37 °C to reach an OD600 of 0.4–0.6, and then isopropyl β-D-thiogalactopyranoside was added to a concentration of 0.2 mM and incubated at 30 °C for 3 h. The cells were lysed in lysis buffer (0.1% Triton X-100, 20 mM Tris–HCl, 200 mM NaCl, and 1 mM EDTA). MBP-β7 was purified from cell lysates using a pMAL Protein Fusion and Purification System (New England Biolabs).
WKY rats (8 week old) were injected intramuscularly at the tail base with an antigen emulsion containing MBP-β7 and Freund’s complete adjuvant (BD Biosciences). Then, 2 weeks later, lymphocytes were collected from iliac lymph nodes and fused with a murine myeloma cell line, SP2/0, using GenomeONE (ISHIHARA SANGYO)33 (link). Hybridoma clones producing mAbs against the active form of β7 were screened by flow cytometry of BAF cells using the hybridoma supernatant in the presence or absence of Mn2+.
WKY rats (8 week old) were injected intramuscularly at the tail base with an antigen emulsion containing MBP-β7 and Freund’s complete adjuvant (BD Biosciences). Then, 2 weeks later, lymphocytes were collected from iliac lymph nodes and fused with a murine myeloma cell line, SP2/0, using GenomeONE (ISHIHARA SANGYO)33 (link). Hybridoma clones producing mAbs against the active form of β7 were screened by flow cytometry of BAF cells using the hybridoma supernatant in the presence or absence of Mn2+.
10
Expression and Purification of MBP-UGT Fusion Proteins
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The pMAL-UGT expression constructs were transformed into E. coli strain Novablue competent cells. An aliquot of 0.3 mM isopropyl-β-D-thiogalactoside was added to induce protein expression when the optical density value of the cell culture (grown at 37 °C) reached 0.5. After 24 h incubation at 16 °C with shaking, the cells were harvested by centrifugation at 4 °C and then stored at −80 °C until purification. The MBP-fusion proteins were purified using maltose binding resin according to the pMAL Protein Fusion and Purification System (New England BioLabs).
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