Magnetic Chromatography brochure - Invitrogen

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A C A pH 3 Lysate (unfractionated) 655 spots pH 3 400 mM fraction Reduced Sample Complexity 802 spots, 68 % unique to this fraction 817 spots, 84 % unique to this fraction DYNAL invitrogen bead separations pH 10 pH 10 B D pH 3 200 mM fraction 867 spots, 84 % unique to this fraction pH 3 pH 10 600 mM fraction Fig. 2. Reduced sample complexity using Dynabeads® WCX A cell lysate from a human cancer cell line (SW480) was fractionated using Dynabeads® WCX. Proteins were adsorbed to Dynabeads® WCX using low salt conditions (50 mM NaP buffer pH 7, 50 mM NaCl) and desorbed step-wise with increasing salt concentrations (50 mM NaP buffer pH 7 including 200 mM, 400 mM or 600 mM NaCl). The figure shows unfractionated lysate (A) and the three fractions (B,C,D) as analysed by 2-D gel electrophoresis. Fraction B, C and D enabled the detection of 2,211 unique resolved spots and of these only 38 spots were found to be present in all three fractions (2%). Analysing the same amount of protein from unfractionated lysate enabled the detection of only 655 spots. Fractions were prepared for gel analysis by desalting using a commercially available 2-D gel clean up kit. Samples were then focused in pH 3 - 10 (11 cm) IPG strips and resolved in 8 - 16 % SDS-PAGE gels. Approximately 100 µg protein was applied to each strip. Gels were silver stained and analysed using a commercially available 2-D gel analysis software. Fig. 3. Reduced sample complexity using Dynabeads® RPC Protein A. Fractionation of a protein mixture containing diamine oxidase and alcohol dehydrogenase using Dynabeads® RPC Protein. The protein mixture (containing 10 μg total protein) was adsorbed to Dynabeads® RPC Protein in 200 mM NaCl, 0.1 % TFA. Salts were subsequently washed away and proteins desorbed step-wise using increasing concentrations of acetonitrile (20 %, 30 % and 40 %). One quarter of each fraction was applied to a 10-20 % SDS-PAGE gel. The gel was silver stained. Diamine oxidase and alcohol dehydrogenase are found in distinct fractions. B. A mixture containing Insulin (1), Ubiquitin I (2), Cytochrome C (3) and Myoglobin (4) (Bruker Daltonics’ Protein Calibration Standard I) was fractionated using Dynabeads® RPC Protein. The mixture was adsorbed to Dynabeads® RPC Protein in 200 mM NaCl, 0.1 % TFA. Salts were subsequently washed away and proteins/ peptides desorbed step-wise using increasing concentrations of acetonitrile (15 %, 30 % and 40 %). An aliquot of protein/peptide mixture or eluate (1 μl) was mixed with an equal amount of MALDI matrix (1 μl sinapinic acid) and 0.5 μl of the sample/matrix mixture spotted onto a MALDI target plate. Data was obtained using a commercially available MALDI-TOF/TOF MS instrument. B Intens. (a.u.) Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] 4 x10 3 2 1 0 x104 2.5 2.0 1.5 1.0 0.5 0 0 x104 2.0 1.5 1.0 0.5 0 0 4000 3000 2000 1000 0 1 pH 10 Unfractionated mixture Pro1_g7_160305\0_G7\1\1SLin Raw 2 3 4 Pro2_g8_160305\0_G8\1\1SLin Raw 15 % acetonitrile fraction Pro3_h6_160305\0_H6\1\1SLin Raw 30 % acetonitrile fraction Pro4_h8_160305\0_H8\1\1SLin Raw 40 % acetonitrile fraction 6000 8000 10000 12000 14000 16000 18000 m/z 3
Intens. (a.u.) Intens. [a.u.] Intens. [a.u.] Intens. Intens. [a.u.] (a.u.) Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] 1500 1000 500 0 x104 3 2 1 0 x104 1.00 0.75 0.50 0.25 x104 1.5 1.0 0.5 4 x10 2 1 8000 6000 4000 2000 0 6000 4000 2000 0 8000 6000 4000 2000 Serum Profiling and Desalting Pro8_g9_160305\0_G9\1\1SLin Raw Untreated sample Pro6_i7_160305\0_I7\1\1SLin Raw Sample desalted using Dynabeads ® RPC Protein 6000 8000 10000 12000 14000 16000 18000 Fig. 4. Desalt your sample pre MS analysis using Dynabeads® RPC Protein A mixture of Insulin, Ubiquitin I, Cytochrome C and Myoglobin containing a high salt concentration (2.2 M NaCl) was desalted using Dynabeads® RPC Protein. The mixture was adsorbed to Dynabeads® RPC Protein, salts were subsequently washed away and proteins/peptides desorbed in 50 % acetonitrile. An aliquot of protein/peptide mixture or eluate (1 μl) was mixed with an equal amount of MALDI matrix (1 μl sinapinic acid) and 0.5 μl of the sample/matrix mixture spotted onto a MALDI target plate. Data was obtained using a commercially available MALDI-TOF/TOF MS instrument. ser2_i4_LMW_040505\0_I4\1\1SLin Dynabeads Raw ® RPC Protein ser4_j3_LMW_040505\0_J3\1\1SLin Dynabeads Raw ® RPC Protein ser5_j5_LMW_040505\0_J5\1\1SLin Dynabeads Raw ® RPC Protein Ser1_l20_110505\0_L20\1\1SLin Dynabeads Raw ® WCX Ser2_m18_110505\0_M18\1\1SLin Dynabeads Raw ® WCX Ser5_n19_110505\0_N19\1\1SLin Dynabeads Raw ® WCX 00 5000 6000 7000 8000 9000 m/z m/z A B Intens. (a.u.) R4_050805_dig buff_TA_L8_100805\0_L8\1\1SRef Raw Fig. 6. Dynabeads® RPC Protein and Dynabeads® WCX can be used for Serum/Plasma Profiling to look for protein (and large peptide) biomarkers A single serum sample was divided into six identical aliquots (10 μl), to which 1 mg of Dynabeads® RPC Protein or Dynabeads® WCX and the appropriate adsorption buffers were added. After washing, proteins (and peptides) were eluted with either 50 % acetonitrile (Dynabeads® RPC Protein) or 0.5 % TFA (Dynabeads® WCX). The results show that even using this non-automated protocol sample preparation is reproducible. Both high and low abundant protein peaks are detected which are above general instrument noise. Since Dynabeads® RPC Protein or Dynabeads® WCX generate different profiles, these beads can be used in parallel on the same serum samples in order to increase the chance of identifying novel biomarkers. An aliquot of eluate (1 μl) was mixed with an equal volume of MALDI matrix (1 μl sinapinic acid) and 0.5 μl of the sample/matrix mixture spotted onto a MALDI target plate. Data was obtained using a commercially available MALDI-TOF/TOF MS instrument. DYNAL invitrogen bead separations 4 Intens. [a.u.] Intens. [a.u.] 5 x10 1.0 0.8 0.6 0.4 0.2 0 0 x105 1.0 0.8 0.6 0.4 0.2 Digest spotted diectly onto MALDI target Pep41_dig buff_E810_TA_G5_100805\0_G5\1\1SRef Raw Digest spotted after clean-up with Dynabeads ® RPC 18 0 0 750 1000 1250 1500 1750 2000 2250 2500 2750 Contaminant Measurement Spotting after Clean-up using Dynabeads ® 6 M Urea RPC 18 Fig. 5. Dynabeads® RPC 18 efficiently remove contaminants from peptide samples As a means of demonstrating desalting efficiency a tryptic Bovine Serum Albumin (BSA) digest (2.5 pmoles) containing 6 M urea, 6 M guanidine, 2 M NaCl or Digestion Buffer* (50 mM NH 4 HCO 3 , 0.5 M urea, 1 mM DTT, 0.1 % TFA) was desalted using Dynabeads® RPC 18. The peptide mixture was adsorbed to Dynabeads® RPC 18, salts were subsequently washed away and peptides desorbed in 50 % acetonitrile. An aliquot of digest or eluate (1 µl) was mixed with an equal amount of MALDI matrix (1 µl HCCA) and 0.5 µl of the sample/ matrix mixture spotted onto a MALDI target plate. Data was obtained using a commercially available MALDI-TOF/TOF MS instrument. An example of typical spectra are shown (A) and the table (B) summarises the efficiency of desalting as demonstrated by sequence coverage, Mascot® Scores and numbers of peptides matched during database searching (n = 6). m/z Direct Spotting onto a MALDI Target Sequence Coverage (%) 35 ± 1 No identification Mascot ® Score 199 ± 7 No identification Peptides Matched 19 ± 1 No identification Sequence Coverage (%) 25 ± 2 No identification Mascot ® 6 M Guanidine Score 142 ± 10 No identification Peptides Matched 14 ± 2 No identification Sequence Coverage (%) 30 ± 8 9 ± 1 Mascot ® 2 M NaCl Score 170 ± 52 37 ± 8 Peptides Matched 16 ± 4 5 ± 1 Sequence Coverage (%) 40 ± 3 No identification Mascot ® Digestion Buffer* Score 238 ± 19 No identification Peptides Matched 22 ± 2 No identification
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Magnetic Chromatography brochure - Invitrogen

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