Bio-Rad Experion Protein Analysis Kits - Manual (Page 15)

3.4 Prepare the Sample Buffers
Separate protein samples under either reducing or nonreducing conditions, but always use reducing
conditions for separation of the Pro260 ladder. Reduced and nonreduced samples can be run on the
same chip. Prepare fresh sample buffer daily.
3.4.1 Reducing Conditions
For each chip, combine 1 µl
β-mercaptoethanol and 30 µl sample buffer (yellow cap). Vortex and spin
down briefly. Protect the sample buffer from light.
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Each chip can analyze up to 10 protein samples. All wells of a chip must be filled for the
electrophoresis station to operate properly.
The total protein concentration, including any added user-defined internal standards, in all starting
samples should be toward the middle of the linear dynamic range of the assay, if possible.
1. Prepare the Pro260 ladder by combining 4 µl Pro260 ladder (red cap) and 2 µl sample buffer with
β-mercaptoethanol (reducing sample buffer) in a microcentrifuge tube.
2. Prepare the samples by combining 4 µl sample and 2 µl sample buffer (Section 3.4) in a
microcentrifuge tube.
3. Vortex all tubes briefly and spin down in a microcentrifuge for a few sec.
4. Heat the samples and Pro260 ladder at 95–100°C for 3–5 min. Spin down the tubes in a
microcentrifuge for a few sec.
5. Add 84 µl deionized water to each tube and vortex briefly to mix. Do not modify this step to adjust
sample concentration.
Both the diluted samples and the Pro260 ladder are stable for several hours when stored at room
temperature and protected from light.
At pH <7, reducing agents such as β-mercaptoethanol and DTT are less effective; use 5–7.5 mM
tributylphosphine (TBP) or tris(2-carboxyethyl)phosphine (TCEP) instead. Otherwise, neutralize the
buffer or use buffers of higher pH before preparing samples for Experion runs.
3.4.2 Nonreducing Conditions
Prepare two stocks of sample buffer: one reducing (for the Pro260 ladder) and one nonreducing
(for the protein samples).
1. For each chip, transfer 30 µl sample buffer (yellow cap) to 2 microcentrifuge tubes.
2. Add 1 µl β-mercaptoethanol to one tube (reducing sample buffer) and 1 µl deionized water to the
other (nonreducing sample buffer). This generates enough nonreducing sample buffer for
10 samples. Vortex the tubes and cover them with foil to protect them from light.
3.5 Prepare the Samples and the Pro260 Ladder
Experion Pro260 Analysis Kit