Escolar Documentos
Profissional Documentos
Cultura Documentos
Every time you use the 2D equipment, you MUST sign the logbook.
Keep all equipment/supplies keratin-free.
Sample Prep
Sample Prep and IEF are absolutely critical for good
results. Almost all problems with 2D can be traced back to these 2
steps especially Sample Prep. Before starting a project – DO YOUR
HOMEWORK!! Read through these guides – between the GE manual
and 3 users, there should be enough information to get you started.
Try to find a paper or someone who has done 2D on your system.
This could help considerably with sample prep. Be careful with
contaminants in your sample/buffers – IEF is very sensitive to
certain chemicals (see GE manual, p. 32, 33). Salt is the most
common sample contaminant. The manual not only describes the
contaminant, it gives the reason it can cause problems AND a
method to remove the contaminant.
The 2D Cleanup kit and 2D Quant kit work well and are HIGHLY
recommended. Your first reaction to using the GE 2D Cleanup kit
and 2D Quant kit is that it is too expensive. But in the long run, it
will save you considerable time and money.
You can get a crude concentration using a spec. or use the BCA
Protein assay kit (Pierce, cat. #: 23227) or a Bradford assay.
Notes:
• Keep sample prep simple to avoid protein loss. It’s a trade-off
you have to decide, additional sample prep may improve the
quality of the final 2-D gel, but at the expense of some protein
loss.
• The cells should be disrupted so that there is minimal
proteolysis. Cell disruption should be done at as low a temp.
as possible and with minimal heat generation. Cell disruption
ideally should be done directly into a strongly denaturing
solution containing protease inhibitors.
• Sample prep solutions should be freshly prepared or stored as
frozen aliquots. Use high purity urea containing protease
inhibitors.
• Prepare the sample just prior to IEF or store samples in
aliquots at –80oC. Do not thaw multiple times.
• Proteins should be kept cold at all times. Do not let sit at RT.
• Remove solid material by centrifugation. Solid particles and
lipids will block the gel pores.
• Do not heat proteins after adding urea. If the soln. is heated
over 30oC, the protein might be modified.
Sample Prep
1. Following is a basic sample prep for 2D gels. Using CyDyes
has additional requirements – see below.
2. Remove cell pellets and lysis buffer (LB) (see Buffers and
Solutions below) from -70C. Place pellets in -20C and allow LB
to thaw at room temp.
3. Vortex LB thoroughly. Making sure all precipitate dissolves.
4. Resuspend the cell pellet in Lysis buffer. Add 1 ml Lysis
buffer/100 ml o/n. Gently rotate tube in your hand. Use a
clean spatula to scrape pellet away from side of tube. Chop
the pellet us as much as you can. No vortexing. You do not
want any frothing now or when you sonicate the sample.
Frothing causes proteins to aggregate.
5. Let sit for 20 min. in Lysis buffer on ice.
6. Sonication.
a. Setup a beaker with ice water with a stir bar in the
bottom and placed on a stir plate. Setup a clamp to hold
the tube in place.
b. Clean sonication tip with 3 rounds of ethanol and water.
c. Following is the program used for Pseudomonas gram (-)
Set total time for 3 min. Pulse 1 sec. on and 0.5 second
off.
d. Stop and swirl/push ice next to tube. As you are pulsing
on and off, turn sonicator dial up slowly. Every 30 sec.,
stop and let sample sit on ice for 1 min.
e. Repeat steps a-e for remaining samples.
7. Spin 10 min. at 12,000 x g. Remove supernatant. Should be
amber color. Transfer lysate to microfuge tube and place on
ice.
8. Remove 100 ul for 2D Cleanup kit. Do a 2D Quant (or other
quantification method) before 2D Cleanup to make sure you
don’t have too much protein. You don’t want more than 100
ug protein/tube. If there is too much protein in the 2D Quant,
the pellet wil be too large to resuspend effectively. Base
Cleanup volume on protein concentration not volume. Do
multiple duplicates of your samples to increase total
concentration and volume.
9. Store remaining protein -80oC.
CyDyes Notes
• CyDyes are VERY sensitive to pH. After 2D cleanup, resuspend
sample in 30 mM Tris, pH 8.8 (pH needs to be between 8.0 and
9.0).
• ABI and Roberto recommendation: If you are doing a big
experiment, you need to randomize your samples and do dye
swapping (Don’t label all controls with Cy3 and all samples
with Cy5 – mix them up). This helps prevent any Dye bias and
aids statistical analysis.
• Running repetitive gels that swap the dyes used to label
samples will control for any dye-specific effects that might
result from preferential labeling or different fluorescence
characteristics of the gel or glass plates at the different
excitation wavelengths of Cy2, Cy3, and Cy5. For example,
one set of gels might use Cy3 to label controls and Cy5 to label
drug-treated samples. A duplicate set of gels should be run
that uses Cy5 to label controls and Cy3 to label drug-treated
samples.
• You also need to do at least 3 biological replicates, not
replicates of the same sample.
• Control and sample variant depend on type of experiment. For
example, control could be uninduced and variant could be
induced; or control could be a 0 time point and variant could
be 5min/10 min/15 min. etc. time point. Whatever your
experiment is - you are comparing 2 conditions on 1 gel. You
would need enough control protein to run with however many
variants you have. For example, if you have 4 time points -
you would run 4 gels with each gel having a 0 time point as
control and 1 time point as variant.
Lysis buffer
30mM Tris, 7M urea, 2M thiourea, 4% CHAPS
20.2 ml 8 M Urea
1 gm CHAPS
4.8 ml dH2O
10.5 gm Urea
3.8 gm thiourea
1 gm CHAPS
bring up to 25 ml with dH2O
Aliquot (2.5 ml) and store for up to 6 months at –20oC.
Put in frig the night before. Thaw on ice until ALL granules are
dissolved. Vortex if needed.
Add 0.5% IPG buffer to whole Destreak bottle solution - 15ul IPG
buffer to 3ml, 60ul IPG buffer to 6ml. Whatever you don’t use –
label bottle with date and whatever pH IPG buffer added. Store -
20oC.
2X sample buffer
2X sample/rehydration solution (use buffer stock 1 or 2, depending
on the rehydration buffer required), 2% Pharmalyte or IPG buffer,
2% (or 20 mg/ml or 130 mM) DTT
40% CHAPS
20 gm CHAPS
Make up to 50 ml with dH2O. Store at –20oC. Stable for 6 months.
Urea/Thiourea solution
8M urea*, 2.3M thiourea
12 gm urea
4.5 gm thiourea
Bring volume up to 25 ml with ddH2O
Add 250 mg amberlite and stir for 1 h
Filter into clean, rinsed bottle.