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Multipep automated peptide synthesizer

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The MultiPep automated peptide synthesizer is a laboratory equipment designed for the automated synthesis of peptides. It performs the necessary chemical reactions and purification steps to produce peptide sequences in a controlled and efficient manner.

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Lab products found in correlation

Rpn1236, by GE Healthcare (1 mentions) Hrp conjugated anti gst antibody, by GE Healthcare (1 mentions) Lasergene software, by DNASTAR (1 mentions) Anti rabbit igg hrp na934v, by GE Healthcare (1 mentions) Ecl prime, by GE Healthcare (1 mentions) Las 1000, by Fujifilm (1 mentions)

8 protocols using multipep automated peptide synthesizer

1

SPOT Synthesis of Peptide Arrays

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SPOT synthesis of peptide arrays on cellulose membranes were performed using a MultiPep automated peptide synthesizer (INTAVIS Bioanalytical Instruments AG, Germany) as previously described [55 (link)]. After blocking the cellulose membranes in TBST with 5% nonfat dry milk, peptide interactions with GST or GST fusion proteins were tested by overlaying the membranes with 1 μg/ml of recombinant protein for 2 hr at room temperature. Filters were washed in TBST, and bound proteins were detected with HRP-conjugated anti-GST antibody (1:5000; clone RPN1236; GE Healthcare).
Joachim J., Razi M., Judith D., Wirth M., Calamita E., Encheva V., Dynlacht B.D., Snijders A.P., O’Reilly N., Jefferies H.B, & Tooze S.A. (2017). Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy. Current Biology, 27(14), 2123-2136.e7.
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2

Peptide Array Analysis of Lamin Proteins

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To generate peptide arrays, human lamin A (P02545, full length), human lamin C (NP_005563, full length), mouse lamin A (P48678, 588-end), and rat lamin A (P48679, 588-end) were synthesized as 20 mer peptides with three amino acid offsets on cellulose membranes using a Multipep automated peptide synthesizer (INTAVIS Bioanalytical Instruments AG, Koeln, Germany) as described [30 (link)]. Peptide array membranes were blocked for 2 h in 1% casein in TBST (Tris-buffered saline with 1% tween) at 22–25 °C, and then incubated overnight at 4°C with a primary antibody (5G4 at 1:2500 dilution; L1283 at 1:2000) in TBST/1% casein. Membranes were then washed three times in TBST (10 min each) and incubated with affinity-purified horseradish-peroxidase-conjugated polyclonal anti-mouse IgG HRP (NA931V) or anti-rabbit IgG HRP (NA934V), both from GE HealthCare (Little Chalfont, UK). Blots were developed using ECL Prime (RPN 2232, GE HealthCare) and chemiluminescence signals were detected using Las 1000 (Fujifilm, Tokyo, Japan). Lasergene software (DNAStar, Madison, WI, USA) was used to generate peptide sequence alignments.
Simon D.N., Wriston A., Fan Q., Shabanowitz J., Florwick A., Dharmaraj T., Peterson S.B., Gruenbaum Y., Carlson C.R., Grønning-Wang L.M., Hunt D.F, & Wilson K.L. (2018). OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A. Cells, 7(5), 44.
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3

Peptide Arrays Probed with Nck Domains

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Peptide arrays were synthesized on nitrocellulose membranes using a MultiPep automated peptide synthesizer (INTAVIS Bioanalytical Instruments AG) as described [46 ]. Membranes were spotted with relevant TSAd peptides and probed with GST-tagged Nck SH2 and SH3 fusion proteins (50–150 μg/ml), followed by anti-GST antibody and anti-mouse HRP secondary antibody. Signals were detected by chemiluminescent detection by Super Signal® West Pico stable peroxide solution (Pierce).
Hem C.D., Sundvold-Gjerstad V., Granum S., Koll L., Abrahamsen G., Buday L, & Spurkland A. (2015). T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells. Cell Communication and Signaling : CCS, 13, 31.
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4

TIPARP Protein Mapping via Peptide Arrays

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The complete sequence of human TIPARP protein (amino acids 1–657; NP_001171646) was synthesized as 20-mer peptides four amino acid offsets on cellulose membranes using a Multipep automated peptide synthesizer (INTAVIS Bioanalytical Instruments, Koeln, Germany) as described previously [31 (link)]. Peptide arrays were blocked in 1% casein in Tris-buffered saline + Tween 20 (TBST) for 1 h at room temperature. The overlays were done using 1 µg/ml GST-TIPARP fusion protein or GST protein in 1% casein TBST overnight at 4°C. Membranes were then washed three times for 10 min in TBST at room temperature. Bound proteins were detected by immunoblotting with 1 : 5000 dilution of anti-GST-HRP (ab58626, Abcam).
Gomez A., Bindesbøll C., Satheesh S.V., Grimaldi G., Hutin D., MacPherson L., Ahmed S., Tamblyn L., Cho T., Nebb H.I., Moen A., Anonsen J.H., Grant D.M, & Matthews J. (2018). Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity. Biochemical Journal, 475(23), 3827-3846.
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5

Mapping ATXN3 N-Terminal Interactions

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The N‐terminal sequence of human ATXN3 (aa 1–182; NP_004984.2) was synthesized as 20‐mer peptides with two amino acid offsets on cellulose membranes using a MultiPep automated peptide synthesizer (INTAVIS Bioanalytical Instruments) as described (Knaevelsrud et al., 2013). Peptide arrays were blocked in 1% casein in PBS‐T for 1 hr at RT. Overlays were done using 1 μg/ml GST‐fusion proteins or GST in 1% casein PBS‐T overnight at 4°C. Membranes were washed three times in PBS‐T and bound proteins detected by immunoblotting with anti‐GST‐HRP.
See Supporting Information Experimental Procedures for all additional experimental procedures.
Herzog L.K., Kevei É., Marchante R., Böttcher C., Bindesbøll C., Lystad A.H., Pfeiffer A., Gierisch M.E., Salomons F.A., Simonsen A., Hoppe T, & Dantuma N.P. (2019). The Machado–Joseph disease deubiquitylase ataxin‐3 interacts with LC3C/GABARAP and promotes autophagy. Aging Cell, 19(1), e13051.
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6

Synthesis and Purification of Arginine-Coupled Peptides

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Peptide arrays were synthesized as described [15 (link)]. In brief, peptide arrays were synthesized on cellulose paper using a Multipep automated peptide synthesizer (INTAVIS Bioanalytical Instruments AG, Koeln, Germany). Arginine coupled peptides were synthesized and purified to 80–95% purity (Genscript Corp, Piscataway, NJ, USA).
Arg9-Syn-4 cyt: R9-RMKKKDEGSYDLGKKPIYKKAPTNEFYA and Arg9 (control peptide): R9. Anti-syndecan-4 (sc-12766) blocking peptide: EGRYFSGALPDDEDVVGPGQESDDFELSG
Rønning S.B., Carlson C.R., Stang E., Kolset S.O., Hollung K, & Pedersen M.E. (2015). Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis. PLoS ONE, 10(6), e0129288.
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7

Peptide Array Synthesis and Probing

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Peptide arrays were synthesized on nitrocellulose membranes using a MultiPep automated peptide synthesizer (INTAVIS Bioanalytical Instruments AG, Germany) as described [26 (link),27 (link)]. Membranes were probed with GST-tagged SH2 fusion proteins (80 μg/ml), and signals were detected using relevant antibodies followed by chemiluminescent detection.
Andersen T.C., Lindsjø K., Hem C.D., Koll L., Kristiansen P.E., Skjeldal L., Andreotti A.H, & Spurkland A. (2014). Solubility of recombinant Src homology 2 domains expressed in E. coli can be predicted by TANGO. BMC Biotechnology, 14, 3.
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8

Peptide Synthesis and Kinase Assay

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Peptides were synthesized as 7- or 15-mers on membranes using a MultiPep automated peptide synthesizer (INTAVIS Bioanalytical Instruments AG, Koeln, Germany). The kinase assay protocol was modified from Himpel et al. [20 (link)]. Membranes were blocked overnight in 15 mL kinase buffer (20 mM Hepes, pH 7.4, 5 mM MgCl2, 1 mM DTT) with 0.2 mg/mL BSA and 100 mM NaCl. Blocked membranes were incubated with 15 mL fresh kinase buffer with 1 mg/mL BSA, 100 mM NaCl and 50 μM cold ATP at 30°C for 45 minutes. Kinase assays were conducted by incubating the membranes in 15 mL of fresh kinase buffer containing 0.2 mg/mL BSA, 12.5 μCi [γ-32P]ATP, and 1 μg of each kinase for 30 minutes at 30°C with slight agitation. Membranes were washed with 15 mL 1 M NaCl for 5 min, for a total of 5 washes, followed by 3 water washes. Membranes were then washed with 15 mL 5% phosphoric acid for 15 min, for a total of 3 washes, followed by 3 water washes. Membranes were air dried, and exposed to film for 3 days. Phosphorylation was analyzed by densitometry using Gilles Carpentier’s Dot-Blot-Analyzer macro for ImageJ (written by Gilles Carpentier, 2008, and available at http://image.bio.methods.free.fr/dotblot.html). See Table S3 for raw data, and normalization protocol.
Lubner J.M., Dodge-Kafka K.L., Carlson C.R., Church G.M., Chou M.F, & Schwartz D. (2017). Cushing’s Syndrome mutant PKAL205R exhibits altered substrate specificity. FEBS letters, 591(3), 459-467.
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