Protocols

ProTocol A: Digestion of Proteins in Solution

Trypsin (SERVA cat. no. 37283, 37284, 37286)

  • Lyophilized Trypsin is reconstituted in 50 mM acetic acid to a final concentration of 1 µg/µl.
  • For digestion of the target protein, add Trypsin to a final protease:protein ratio of 1:100 to 1:20 (w/w).

Endproteinase Glu-C (SERVA cat. no. 20984)

  • Digestion buffer: 100 mM NH4HCO3, pH 7.8 (optional: 100 mM Tris/HCl, pH 7.8)
  • Reconstitute the lyophilized enzyme in deionized water to make a 100 ng/μl solution.
  • Prepare a 5 ng/μl enzyme in digestion buffer by combining 5 μl of the 100 ng/μl enzyme solution with 95 μl digestion buffer.
  • The specific volumes and concentration can be adjusted for sample numbers and/or quantity of protein in the sample.
  • A final enzyme to protein ratio of 1:20 to 1:100 is recommended.

Lysyl-Endproteinase® (Lys-C, SERVA cat. no. 20987)

  • Digestion buffer: 50 mM Tris/HCl, pH 8.5
  • Reconstitute the lyophilized enzyme in digestion buffer to make a 1 ng/μl solution.
  • A final enzyme to protein ratio of 1:20 to 1:100 is recommended.

ProTocol B: In-gel Protein Digestion

Trypsin (SERVA cat. no. 37283, 37284, 37286)

  • Lyophilized Trypsin is reconstituted in 50 mM acetic acid to a final concentration of 1 µg/µl.
  • Add 25 mM NH4HCO3, pH 8 to get a final Trypsin concentration of 50 µg/ml.
    For example: 25 µl Trypsin solution (1 µg/µl) + 475 µl NH4HCO3, pH 8 (25 µg Trypsin/500 µl = 50 µg Trypsin/ml)
  • For the final use dilute the Trypsin solution 1:2.5 with 25 mM NH4HCO3, pH 8 and use 10 – 20 µl for rehydration/digestion of each gel piece.

Endproteinase Glu-C (SERVA cat. no. 20984)

  • Digestion buffer: 100 mM NH4HCO3, pH 7.8 (optional: 100 mM Tris/HCl, pH 7.8)
  • Combine 10 μl of the 100 ng/μl reconstituted enzyme with 90 μl of digestion buffer to achieve a final concentration of 10 ng/μl. Place on ice until ready to use.
  • Add 50 μL of the 10 ng/μl enzyme solution to the dried gel pieces and incubate on ice for 45 min.
  • Remove the 10 ng/μl enzyme solution from the gel pieces with a micro pipettor and replace with 50 μl of digestion buffer.
  • Incubate overnight at 37 °C.

Lysyl-Endproteinase® (Lys-C, SERVA cat. no. 20987)

  • Digestion buffer: 50 mM Tris/HCl, pH 8.5
  • Reconstitute the enzyme in digestion buffer to achieve a final concentration of 10 ng/μl.
  • Add 100 μL of the enzyme solution to the dried gel pieces and incubate on ice for 45 min.
  • Remove the enzyme solution from the gel pieces with a micro pipettor and replace with 10 μl of digestion buffer.
  • Incubate overnight at 37 °C.

Peptide Extraction (Recommended to avoid clogging of the LC system)

  • Clear solution of the In-Gel digest by centrifugation and transfer the supernatant into a new vial.
  • Alternative method: Extraction of the peptides, e.g. with acetic acid and actonitrile.

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SERVA Catalog
2022

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Western Blotting -
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New !

SERVA Catalog
2022

In detail you will find the following new products in catalog 2022:

New glycosidases such as Endo F3, Endo S and Sialidase
Blotting kits to match the "Easy Peel" 2D HPE™ gels for blotting horizontal 2D gels
New protein standards, e.g. SERVA Triple Color Protein Standard I    and the SERVA Fluo-610 Protein Standard I, pre-stained for direct fluorescence detection
BlueVertical™ PRiME™ blot module and gel casting stand for SERVA's BV-104   electrophoresis  tank
he albumin portfolio has been expanded with the "Albumin bovine Fraction V, protease- free, low IgG"
Ready-to-use CL HRP Western Blotting Substrates SERVALight "PreMix"

 

SERVA Catalog 2022.pdf
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Now click on the search result and you will be taken to the product entry. Here you will find the link "Certificate of Analysis", which you follow.
Now select the lot number from the list and download the PDF with a click. Done.

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Western Blotting -
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  • • Molecular Weight Standards
  • • SDS PAGE for Western Blotting
  • • Transfer Membranes and Buffers
  • • Blocking Reagents
  • • Detection Systems
  • • Instruments for Western Blotting
SERVA Brochures: Western Blotting V2110 (pdf)
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