User Manual

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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