2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:51 PM Page 44
Technical Appendix
Brief Guidelines for Freezing and Thawing Protein Samples
Disclaimer: These guidelines are intended for use in general with • Allow the liquid to cool until it has a syrupy consistency.
protein solutions, but the stability of individual proteins varies widely. • Place tubes in the rack allowing them to snap freeze in the covered
The investigator must determine the proper storage and freeze-thaw ice bucket.
conditions for each protein. • Once tube contents are frozen, rapidly wipe the liquid off (blot, do not
wipe the label – alcohol can solubilize ink), transfer tubes to a pre-
For proteins purchased from PBL InterferonSource, refer first to the data chilled box (possibly in another bucket with dry ice (no liquid!), then
sheet(s). In the absence of written guidance, contact technical service place tubes in a -80ºC freezer.
(at +1-732-777-9123) for recommendations on handling of specific • After the freeze, allow all the dry ice to evaporate in the fume hood
proteins. and the alcohol to warm to room temperature.
It is usually best to work with protein solutions on wet ice. The low • Decant the remaining isopropanol into a bottle labeled according to
temperature will slow inactivation of the protein. OSHA and State labeling standards with the additional qualifier of
Never vigorously agitate a protein sample. The preferred method of “Freezing Isopropanol.” This isopropanol can be reused for freezing
mixing protein solutions is to gently mix with a micropipettor using a many times.
polypropylene tip (Do not use larger pipettors with polystyrene pipettes).
An alternative for larger solutions is gentle inversion in a 15 to 50 ml Alternative
capped polypropylene tube. A slightly less effective way to freeze the samples is simply to prepare
For volumes over 50 ml, mixing using a stir bar is acceptable, but not a bucket of dry ice, then insert the tubes into the dry ice until they are
too vigorously. Never introduce foam or air bubbles as these denature almost buried. This will freeze the small aliquots sufficiently quickly,
proteins. but freezing of larger volumes/tubes will take longer, allowing loss of
Do not use glass or polystyrene containers or pipettes for antibodies homogeneity in the samples.
or interferons unless these proteins are diluted in serum or albumin-
containing media. Thawing Method
Freeze/Thaw of proteins. Usually a quick freeze and a quick thaw are • The preferred method of thawing protein samples is to place the tube
the best methods for retaining protein activities. Rapid freezing and in cold tap water. Remove the tube from the freezer, wipe off the rim
thawing prevent phase partitioning of the salts or protein. If a protein and then place it in a beaker with some cold tap water. “Floaties” used
solution is found to be cloudy upon thawing, the bioactivities of the in the water baths work well for multiple samples.
protein are likely to have been adversely affected. • Once thawed, gently mix the sample using a micropipettor to make
sure the solution is homogeneous. Again, be careful not to introduce
Freezing Method bubbles into the solution.
Preferred • Place the thawed tube on wet ice.
• Be aware that glass tubes can crack, leak, or explode upon snap • Check the protein solution for cloudiness.
freezing or thawing of solutions. Polypropylene tubes are • Very small samples such as 10 µl aliquots can be thawed by holding
recommended for snap freezing. the tube in your hand and monitoring thawing.
• Prior to freezing, make sure the solution identification number is
written on the top of each tube and/or on a freezer-safe label using an Avoid introducing bubbles or foam into any protein solution!
alcohol-resistant marker.
• Always take appropriate precautions when working with dry ice. Most proteins are unstable at very dilute concentrations in the absence
Handle only with appropriate tools and gloves. Never touch dry ice or of carrier. Unless formulation work has been done to verify stability, do
its solutions with bare hands. Refer to the MSDS sheet for dry not dilute carrier-free solutions to protein concentrations below 100
ice/solid CO
2
. Also, when working with dry ice and its solutions, be µg/ml. This is only a guideline and needs to be verified for each protein.
certain to work in a well ventilated area, preferably in a chemical Be aware that all interferons will stick somewhat to plastics. When
fume hood where CO
2
gas will not accumulate. performing serial or multiple dilutions, change pipette tips between
• Set up a tube rack in an ice bucket. each dilution step. Rinsing the pipette tip is insufficient to prevent
• In the ice bucket, surround the rack with dry ice and then add carryover between wells of tubes. Interferon beta protein tend to exhibit
sufficient isopropanol to saturate the dry ice. Cover with an ice greater hydrophobicity than most interferon alpha proteins, thus more
bucket lid. prone to “stickiness”.
PBLInterferonSource Toll Free: 1 877- PBL-8881
44
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