C.B.S. Scientific HTLE-7002 - Manual (Page 11)

C. B. S. œ Scientific 11 HTLE-7002
3.2 Preparation of Samples-continued
A. Elution from Polyacrylamide Gels--continued
Remove the tube and incubate at 37
°C or RT for at least 90min with shaking (we sometimes
let this step go overnight). Next, the suspension is vortexed briefly and then centrifuged for
5-10min at low speed (2-3,000 rpm) in a swinging bucket table-top centrifuge to pellet the gel
bits. Carefully remove the supernatant using a disposable plastic transfer pipet (avoiding any
debris), then place it in a new 1.5ml microcentrifuge tube and measure its volume. The
disposable micro-transfer pipets from Research Products International Corp. are particularly
good for this purpose because they have a very narrow and drawn-out tip. A second volume
of ammonium bicarbonate plus SDS and
β-mercaptoethanol is added to wash the gel bits
and to further elute any residual protein. Since the volume of total eluate collected should be
roughly 1.2ml, the second volume of buffer to be added should equal 1.2ml minus the volume
of the first supernatant. After adding the second volume of elution buffer, replace the cap
and vortex the tube briefly. You may also reheat the suspension for another 3-5min
(optional) before incubating again at 37
°C for 30-60min, or at room temperature for at least 2
hours. The elution period, steps 1 and 2 combined, should be at least 1.5hr and may go
overnight. After the second elution step the gel bits are removed again by centrifugation, and
the second supernatant is removed and combined with the first (should be about 1.2ml).
Clarify the total eluate by spinning in a microfuge for 5min to pellet any residual debris, then
carefully remove the supernatant to another new tube. It is essential that no particulate
debris remain in the clarified supernatant. Place the tube on ice and chill.
Elution from polyacrylamide gel can also be accomplished by electrophoretic transfer of
proteins to nitro-cellulose, nylon or PVDF membranes (western blot) (1, 2). Labeled proteins
directly transferred to membranes can be visualized and excised using the same method
outlined above. Instead of eluting protein from a gel piece, the membrane fragment
containing the labeled protein or peptide is incubated directly with trypsin and the tryptic
peptides are released in a soluble form(1). This eluate is then lyophilized and washed, and
the peptides ready for analysis (see SECTION 3.3 below).
B. Precipitation
To concentrate the labeled protein and to remove any SDS, the proteins are precipitated
using tricloroacetic acid (TCA) then washed with a volatile organic solvent. The proteins are
then completely oxidized in the presence of performic acid. This step will fully oxidize all
methionine and cysteines residues present so they exist in a single oxidation state
(methionine sulfone and cysteic acid respectively). This will prevent artefactual separation of
oxidation isomers during chromatography.
At some point during the TCA precipitate step, you should prepare the performic acid by
adding 900
μl of formic acid (∼98%) and 100μl of hydrogen peroxide (33%) to a 1.5ml
Eppendorf tube. Performic acid is formed during incubation at RT for 30-60min, then chill by
placing on ice.
The eluted protein is precipitated in he presence of carrier protein and 15-20% TCA. We
often use heat-treated RNase A because it is both a good carrier and it can degrade
contaminating RNA. However, other proteins such as IgG and BSA can be used as well. Add
20
μg of carrier (RNase A) to the chilled eluate, followed by 250μl of ice cold TCA (100%). Mix
well, then leave on ice for at least 60min. Collect the TCA precipitate by centrifugation in a
micro-microfuge for 10min at 4
°C. Carefully decant the supernatant into a new tube, leaving
the final 50-100
μl behind (you may not see the pellet at this stage). Recentrifuge and remove
the remaining supernatant, combining it with the first TCA supernatant. Wash the precipitate
with 500
μl of either absolute ethanol or acetone (ice-cold), and centrifuge again for 5min,
4
°C. Remove the supernatant, doing 2 spins as above for TCA. The pellet should be obvious
at this stage. Air dry the pellet (do not lyophylize).