Macrospin columns

Manufactured by Harvard Apparatus

Sourced in United States

Macrospin columns are a type of laboratory equipment used for the purification and separation of biological samples. They feature a spin column design that allows for efficient and rapid sample processing. The core function of Macrospin columns is to facilitate the separation and isolation of target molecules, such as proteins, nucleic acids, or other biomolecules, from complex mixtures.

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Lab products found in correlation

Sequencing grade lysyl endopeptidase, by Fujifilm (5 mentions) Sequencing grade trypsin, by Promega (4 mentions) Trypsin, by Promega (4 mentions) Iodoacetamide, by Merck Group (3 mentions) Microspin, by Harvard Apparatus (2 mentions) Microspin columns, by Harvard Apparatus (2 mentions) Ammonium bicarbonate, by Promega (2 mentions) Tris 2 carboxyethyl phosphine, by Merck Group (2 mentions) Ammonium bicarbonate, by Merck Group (2 mentions) Ammonium bicarbonate buffer, by Merck Group (1 mentions) Dithiothreitol (dtt), by Roche (1 mentions) Ms grade trypsin, by Promega (1 mentions) Trifluoroacetic acid tfa, by Merck Group (1 mentions) Immobiline drystrip ph 3 10, by GE Healthcare (1 mentions) 3100 offgel fractionator, by Agilent Technologies (1 mentions) Tissuelyser, by Qiagen (1 mentions) Urea, by Merck Group (1 mentions) Lys c, by Fujifilm (1 mentions) Rapigest, by Waters Corporation (1 mentions) Easy nlc 2, by Thermo Fisher Scientific (1 mentions)

15 protocols using macrospin columns

Proteome Analysis of Phages fEV-1 and fD1

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For the proteome analyses, the phages fEV-1 and fD1 were purified as described above, except that the final resuspension after ultracentrifugation was done in SM buffer without sucrose. The purified fEV-1 phage preparation was digested for mass spectrometry in triplicates, whereas the fD1 dataset consisted of two different phage preparations both as triplicate samples. Briefly, the samples were mixed with 8 M urea and 100 mM ammonium bicarbonate, and the cysteine bonds were reduced with 5 mM TCEP (37 °C for 30 min) and alkylated with 10 mM iodoacetamide (22 °C for 60 min). Samples were diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M, and sequencing grade trypsin (Promega, Madison, WI, USA) was added for protein digestion (37 °C for 18 h). Samples were acidified (to a final pH 3.0) with 10% formic acid, and the peptides subsequently purified with C18 reverse phase spin columns, according to the manufacturer’s instructions (Microspin and Macrospin columns, Harvard Apparatus, Holliston, MA, USA). Peptides were dried using a Speedvac and reconstituted in 2% acetonitrile/0.2% formic acid prior to mass spectrometric analyses.

Skurnik M., Jaakkola S., Mattinen L., von Ossowski L., Nawaz A., Pajunen M.I, & Happonen L.J. (2021). Bacteriophages fEV-1 and fD1 Infect Yersinia pestis. Viruses, 13(7), 1384.

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Protein Digestion and Purification Protocol

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The samples from AP and plasma adsorption experiments were mixed with 8 M urea and 100 mM ammonium bicarbonate, and the cysteine bonds were reduced with 5 mM TCEP (37 °C for 30 min) and alkylated with 10 mM iodoacetamide (22 °C for 60 min). Samples were diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M, and sequencing grade trypsin (Promega) was added for protein digestion (37 °C for 18 h). Samples were acidified (to a final pH 3.0) with 10% formic acid, and the peptides subsequently purified with C18 reverse phase spin columns according to the manufacturer’s instructions (Microspin and Macrospin columns, Harvard Apparatus). Peptides were dried in a speedvac and reconstituted in 2% acetonitrile, 0.2% formic acid prior to mass spectrometric analyses.

Happonen L., Hauri S., Svensson Birkedal G., Karlsson C., de Neergaard T., Khakzad H., Nordenfelt P., Wikström M., Wisniewska M., Björck L., Malmström L, & Malmström J. (2019). A quantitative Streptococcus pyogenes–human protein–protein interaction map reveals localization of opsonizing antibodies. Nature Communications, 10, 2727.

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Protein Denaturation and Digestion Protocol

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Samples from cross-linking of purified proteins and cross-linking of plasma adsorption were denatured in 8 M urea–100 mM ammonium bicarbonate, and the cysteine bonds reduced with 5 mM tris(2-carboxyethyl)phosphine (37 °C, 30 min) and alkylated with 5 mM iodoacetamide (22 °C, 60 min). Samples were diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M, and sequencing-grade lysyl endopeptidase (37 °C, 2 h) (Wako Chemicals) followed by trypsin (37 °C, 18 h) (Promega) was added for protein digestion. Digested samples were acidified with 10% formic acid to a pH of 3.0, and the peptides were subsequently purified with C18 reverse-phase spin columns according to the manufacturer’s instructions (Macrospin columns, Harvard Apparatus). Dried peptides were reconstituted in 2% acetonitrile and 0.2% formic acid prior to MS analyses.

Hauri S., Khakzad H., Happonen L., Teleman J., Malmström J, & Malmström L. (2019). Rapid determination of quaternary protein structures in complex biological samples. Nature Communications, 10, 192.

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Disulfide Bond Reduction and Alkylation

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Reduction and alkylation of disulfide bonds on proteins were carried out using 1 M dithiothreitol (Roche, Switzerland; final sample concentration 10 mM) for 45 min and 1 M Iodoacetamide (Sigma-Aldrich; final sample concentration 30 mM) for 30 min in a dark, respectively. Following alkylation and reduction, the samples were diluted with ammonium bicarbonate buffer (pH 8.0) until the urea concentration was 1 M (Sigma-Aldrich). The proteins were digested with trypsin (MS grade; Promega, Madison, WI, United States) overnight at 37°C at an enzyme to protein ratio of 1:20. Finally, the peptides were acidified with 100% Trifluoroacetic acid (TFA; Sigma-Aldrich) to a final concentration of 1% TFA and then desalted using macro spin columns (Harvard apparatus, Holliston, MA, United States).

Magnusson R., Rundquist O., Kim M.J., Hellberg S., Na C.H., Benson M., Gomez-Cabrero D., Kockum I., Tegnér J.N., Piehl F., Jagodic M., Mellergård J., Altafini C., Ernerudh J., Jenmalm M.C., Nestor C.E., Kim M.S, & Gustafsson M. (2022). RNA-sequencing and mass-spectrometry proteomic time-series analysis of T-cell differentiation identified multiple splice variants models that predicted validated protein biomarkers in inflammatory diseases. Frontiers in Molecular Biosciences, 9, 916128.

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Protein Reduction, Alkylation, and Trypsin Digestion

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First reduction and alkylation of disulfide bonds on proteins were carried out using 1 M DTT, final sample concentration 10 mM, for 45 min and 1 M IAA, final sample concentration 30 mM, for 30 min in the dark, respectively. Following alkylation and reduction the samples were diluted with ammonium bicarbonate buffer (pH 8.0) until the urea concentration was 1 M. The proteins were digested with trypsin overnight at 37°C at an enzyme to protein ratio of 1:20. Finally, the peptides were acidified with 100% trifluoroacetic acid (TFA) to a final concentration of 1% TFA and then desalted using macro spin columns (Harvard apparatus, USA).

Zenere A., Rundquist O., Gustafsson M, & Altafini C. (2022). Multi-omics protein-coding units as massively parallel Bayesian networks: Empirical validation of causality structure. iScience, 25(4), 104048.

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Off-Gel Electrophoresis Sample Preparation

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Before OGE, samples were dried under speed-vacuum then purified by using Macrospin columns (Harvard Apparatus, Holliston, US-MA) according to manufacturer's recommendations. Tubes were dried under speed-vacuum and A 3100 OFFGEL Fractionator (Agilent technologies, Santa Clara, US-CA) was performed over night to separate the sample. Guidelines available in Agilent datasheet were followed, using a 13cm IPG strip (Immobiline DryStrip pH 3-10, 13cm GE Healthcare, Little Chalfont, UK) and 12 OGE wells (20, 21) . After fractionation, microspin columns (Harvard Apparatus, Holliston, US-MA) were used according to the manufacturer's recommendations and the 12 fractions of each experiment were dried under speed-vacuum. Peptide concentration of the fractions was theoretically approximated, considering that 1/12 of the pooled sample was found in each fraction after OGE.

Dor M., Eperon S., Lalive P.H., Guex-Crosier Y., Hamedani M., Salvisberg C, & Turck N. (2019). Investigation of the global protein content from healthy human tears. Experimental eye research, 179.

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Quantitative Proteomics of Cra Regulator

Cited 2 times

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The wild-type and cra deletion mutant harboring the IPTG-inducible plasmid
pP_tac-cra were grown in M9 medium plus glucose (5 g l⁻¹) or M9 plus
acetate (0.75 g l⁻¹) plus 0 or 10 μM IPTG. Harvested cells were washed and
lysed, and the extracted proteins were digested with trypsin as described previously (Malmstrom
et al, 2009 (link)). Then, 10 pmol of heavy labeled
reference peptide was added to the digests, each containing 100 μg of total peptide. After
desalting the peptides with macro-spin columns (Harvard Apparatus), an aliquot containing 1
μg of peptide was subjected to targeted mass spectrometry using previously specified
instrument settings (Picotti et al, 2009 (link)).
The total number of cells in the samples was determined by flow cytometry. Averages and standard
deviations were derived from glucose experiments for the wild-type cells and from glucose and
acetate experiments for other strains.

Kotte O., Volkmer B., Radzikowski J.L, & Heinemann M. (2014). Phenotypic bistability in Escherichia coli's central carbon metabolism. Molecular Systems Biology, 10(7), 736.

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Peptide Fractionation by pI Separation

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Previously dried samples were resuspended in 5% CAN and 0.1%FA and purified under Macrospin columns (Harvard Apparatus, Holliston, MA). A 3100 OFFGEL Fractionator (Agilent Technologies, Les Ulis, France) was then used to separate peptides according to their pI, as reported previously^{16 (link)} with a 13 cm IPG strip (immobiline Dry strip pH 3–10, 13 cm; GE Healthcare, Little Chalfont, UK) and 12 OGE wells. The focusing parameters were 20 Kvh, 800v, 50 uA, 200 mW, and 100 s. The hold parameters were 500 V, 20 uA, and 50 mW. After overnight fractionation, microspin columns (Harvard Apparatus) were performed according to manufacturer's recommendations and the 12 fractions were dried under speed vacuum.

Kowalczuk L., Matet A., Dor M., Bararpour N., Daruich A., Dirani A., Behar-Cohen F., Thomas A, & Turck N. (2018). Proteome and Metabolome of Subretinal Fluid in Central Serous Chorioretinopathy and Rhegmatogenous Retinal Detachment: A Pilot Case Study. Translational Vision Science & Technology, 7(1), 3.

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Purification of MHCII Complexes from Tissue Lysates

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The purification of MHCII complexes from tissue lysates was essentially
performed as described (7 (link)). Due to
enlargement of mesenteric LNs of animals with chemically-induced colitis and
variation in the weight of these LNs, 20 to 25 mg of total tissue were pooled
for the analysis of inflamed LNs, corresponding to one to two LNs. In brief, LNs
were disrupted with a tissue lyser (Quiagen) for 2 mins at 25 Hz in lysis buffer
(0.5% deoxycholate, 1% Igepal CA-630, 50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.2 mM
iodoacetamide, 1 mM EDTA, 1 mM PMSF, Roche Complete Protease Inhibitor Cocktail
pH 7.4). MHCII complexes were purified from tissue lysates with M5/114 antibody
coupled to solid support. After a two hour incubation at 4°C, unbound
protein was removed by washing with 10 ml of lysis buffer, 10 ml of buffer A
(150 mM NaCl, 20 mM TrisHCl pH 7.4), 10 ml of buffer B (400 mM NaCl, 20 mM
TrisHCl pH 7.4), 10 ml of buffer A, and 10 ml of buffer C (20 mM TrisHCl pH
8.00). MHCII complexes were eluted after the last wash in 1 ml of 10% acetic
acid. Eluates were subjected to C18 purification using Macro SpinColumns
(Harvard Apparatus). Peptides were eluted in 25% acetonitrile, 0.1%
trifluoroacetic acid, and dried with a vacuum concentrator (Christ Alpha RVC).
The dried peptides were stored at -20°C until MS analysis.

Fugmann T., Sofron A., Ritz D., Bootz F, & Neri D. (2016). The MHC class II immunopeptidome of lymph nodes in health and in chemically-induced colitis. Journal of immunology (Baltimore, Md. : 1950), 198(3), 1357-1364.

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Sample Preparation for Mass Spectrometry

Cited 2 times

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The sample preparation for mass spectrometry was done as described [4 (link),15 (link)]. Briefly, the samples were denatured in 8 M urea-100 mM ammonium bicarbonate (both Sigma), and the cysteine bonds reduced with 5 mM tris(2-carboxyethyl)phosphine (Sigma) (37 °C, 30 min). The cysteines were alkylated with 5 mM iodoacetamide (Sigma) (22 °C, 60 min), and the samples subsequently digested using sequencing-grade lysyl endopeptidase (37 °C, 2 h) (Wako). The samples were diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M and followed by digestion using trypsin (Promega) (37 °C, 18 h). Digested samples were acidified with 10% formic acid to a pH of 3.0, and the peptides were subsequently purified with C18 reverse-phase spin columns according to the manufacturer’s instructions (Macrospin columns, Harvard Apparatus). Dried peptides were reconstituted in 2% acetonitrile and 0.2% formic acid prior to MS analyses.

Khakzad H., Happonen L., Karami Y., Chowdhury S., Bergdahl G.E., Nilges M., Tran Van Nhieu G., Malmström J, & Malmström L. (2021). Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology. PLoS Computational Biology, 17(1), e1008169.

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