C.B.S. Scientific TTGE-2401 - Manual (Page 3)

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SAMPLE PREPARATION
Sample Preparation - Prepare samples using ClearPAGE sample buffers for
optimal resolution. Other buffers can be used but resolution will suffer. 1 part
ClearPAGE 4x LDS sample buffer can be added to 5 parts existing sample to aid
with loading and stacking into the gel.
1) For DNA/Native Gels, ClearPAGE 4x DNA/Native Sample Buffer (Cat #
GB33002) is required for sample preparation.
2) For SDS Gels, ClearPAGE 4x LDS Sample Buffer (Cat # FB31002/FB31010)
is required for sample preparation.
a) Allow Sample Buffer to equilibrate to room temperature
b) For reducing conditions
i) Combine Sample and ultrapure water (if necessary) to achieve 65% of
the final volume
ii) Add 25% of the final volume of 4x LDS Sample Buffer
iii) Add 10% of the final volume of reducing agent (e.g., Cat # FB32001
DTT Reducing Agent 10x).
iv) Heat the samples for 10 minutes at 70°C (preferred) or 3 minutes at
100°C.
v) Once reduced, run samples within 2 hours to prevent reoxidation.
c) For non-reducing conditions
i) Combine Sample and ultrapure water (if necessary) to achieve 75% of
the final volume
ii) Add 25% of the final volume of 4x LDS Sample Buffer (Cat #
FB31002/FB31010)
iii) Heat the samples for 10 minutes at 70°C (preferred) or 3 minutes at
100°C.
Below is an example on how to prepare 100 μl of 1 mg protein/ml running
sample from a 5 mg protein/ml initial sample.
Solution Reduced Not Reduced Well Type Max. Volume 40% Max. Vol.
Sample 20μl 20μl 12-well 35μl 14μl
Water 45μl 55μl 17-well 17μl 6.8μl
Sample Buffer 25μl 25μl
1 Prep
well
400μl 280μl
Reducing
Agent
10μl None 2D-well 300μl 210μl
GEL PREPARATION
1) The comb has been removed from these gels prior to shipping and there is
also no tape to remove. Just before use remove the gel cassette from it’s plastic
storage bag and shake gel off upside down.
2) Rinse entire gel cassettes using ultrapure water with a wash or squeeze bottle.
3) Rinse wells two times using ultrapure water with a wash bottle, shake to
remove water from wells after each wash.
LOADING SAMPLES ONTO THE GEL:
1) To load samples slide precast gel cassette into core assembly so that the
notched plate faces towards the core upper reservoir.
2) After rinsing wells, fill with ultrapure water. Density differential between
water and sample buffer aids protein stacking into the gel. If filling with
running buffer, leaving for an extended period will adversely effect resolution.
3) When running one gel, use white plastic adaptor plate to seal the other side.
Close core doors and press down on white latches. Fill upper core reservoir