Bio-Rad ProteoMiner Protein Enrichment Kits - Manual (Page 19)

Section 11
Appendix
Preparation for Various Downstream Applications
Prior to any downstream analysis, you will need to quantitate the amount of
protein in your sample. For this we recommend the Quick Start™ Bradford
protein assay (Catalog #500-0201). If your downstream analysis technique is
negatively impacted by low pH or salts, we recommend that you clean up your
sample using Bio-Rad's ReadyPrep™ 2-D cleanup kit (catalog #163-2130).
2-D gel electrophoresis users : The reconstituted elution reagent used
to elute your sample is acidic. If using DIGE, adjust pH of eluent to
approximately 8.5 with 4 M sodium carbonate. Add approximately 30 µl of 4 M
sodium carbonate to 300 µl eluent to bring pH up to 8.5. It is recommended
that you then remove excess salts using Bio-Rad's ReadyPrep 2-D cleanup kit
(catalog #163-2130) prior to loading sample on an IPG strip. Alternatively,
proteins may be eluted by replacing the elution reagent with lysis buffer
(25 mM Tris, 4% CHAPS (w/v), 8 M urea, 2 M Thiourea). However, this may
result in a decreased number of protein spots.
Loading samples on IPG strips : The total amount of protein to load
per strip will vary depending on the sample, the pH range, length of the IPG
strip, and the detection system used. In some cases, overloading of protein is
acceptable to reveal less abundant proteins of interest. Below is a guideline for
protein loads that generally gives acceptable 2-D patterns. Use lower amounts
for silver or SYPRO Ruby protein staining and higher amounts for Coomassie
Blue staining. In general, the maximum that can be loaded onto an IPG strip is
500 µg for 7 cm, 1 mg for 11 cm, 3 mg for 17 cm/18 cm, and 4 mg for
24 cm.
Recommended Range of Protein Loads for IPG Strips
IPG strip length 7 cm 11 cm 17 cm 18 cm 24 cm
Rehydration volume
per strip 125 µl 200 µl 300 µl 315 µl 450 µl
Protein load
Silver stain
5–20 µg 20–50 µg 50–80 µg 50–80 µg 80–150 µg
Protein load
Coomassie Blue G-250 50–100 µg
100–200 µg 200–400 µg 200–400 µg 400–800 µg
For SELDI analysis : The sequential elution buffers allow for direct use of
samples for SELDI analysis. Use a 1:10 dilution of the extracted sample in the
appropriate ProteinChip
® binding buffer and incubate the sample for 1 hr with
shaking. For on-spot assays, use 0.5 µl of extract and 4.5 µl of the
appropriate binding buffer. For Bioprocessor assays, use 5.0 µl of extract and
45.0 µl of appropriate chip binding buffer.
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