C.B.S. Scientific HTLE-7002 - Manual (Page 26)

C. B. S. œ Scientific 26 HTLE-7002
3.4 Second Dimension: Thin Layer Chromatography
The second dimension is thin layer chromatography using an organic solvent buffer to separate
peptides based on their ability to partition between the mobile (buffer) and stationary (cellulose)
phases. We use large glass tanks (C.B.S. cat. # LCT-100) that were traditionally used for
descending paper chromatography (52 X 23 X 56cm). One can also use the smaller glass “brick”
tanks instead if they are equilibrated with buffer using Whatman 3MM paper to line three sides of
the tank up to the top. The chromatography tanks should be kept in a quiet area of the lab or in a
separate room to avoid excess vibrations that disturb chromatography. Buffers should be made
in advance of the chromatography step and allowed to equilibrate completely before use. Make
sure that the lid forms a tight seal with the tank using silicone or vacuum grease.
A. Choice and Preparation of Chromatography Buffers
We use three different chromatography buffer systems and choose the appropriate buffer
depending on the type of separation required. They are: I) Regular Chromatography buffer is
used for maps of proteins with containing peptide with solubilities ranging from very hydrophilic
to very hydrophobic. When performing total protein fingerprints, such as for S
35
-methionine
labeled or for I
125
-iodinated proteins, Regular Chromatography buffer is probably the best choice.
ii) Phosho-Chromatography buffer contains more aqueous solvents, and as its name implies, is
good for separating relatively hydrophilic peptides such as phosphopeptides. iii) Isobutyric Acid
buffer is the most aqueous of the three and is used in special cases to resolve extremely
hydrophilic peptides. Unfortunately there are no strict rules which one can apply to help predict
which buffer to choose. One will probably have to test the migration patterns using each buffer
system to analyze a given protein. It is probably best to begin using Regular Chromatography
buffer for metabolically labeled or iodinated proteins and Phospho-Chromatography buffer for
phosphopeptide analysis.
For 2.01 of each buffer:
Regular Chromatography Buffer
n-butanol
785ml
pyridine 607ml
acetic acid 122ml
deionized water 486ml
Phospho-Chromatography Buffer
n-butanol
750ml
pyridine 500ml
acetic acid 150ml
deionized water 600ml
Isobutyric Acid Buffer
isobutyric acid
1251ml
pyridine 96ml
acetic acid 58ml
n-butanol 38ml
deionized water 558ml