Bio-Rad Silver Stains - Manual (Page 7)

Problem Solution
Some proteins are not staining. Silver stain as usual, then recycle gel.
Soak in deionized water for 30 minutes,
then repeat the silver staining starting with
the silver reagent step (step 8). Proteins
that did not stain on the first cycle will
stain to full intensity
Negative staining occurring. Proteins are overloaded. Recycle gel as
above. Some metalloproteins will always
stain negatively.
Silver reagent is being washed away.
Decrease time of wash in step 9.
Contaminant bands across Skin keratin (from dandruff, ungloved hands)
entire gel, occurring at 58 kD contaminating solutions. Wear gloves at all
and 64 kD. times during the pouring and running of
gels and solution preparation. Filter all
solutions through a 0.45 µm nitrocellulose.
Large discolored spots on gel. Any pressure on gel will cause the gel to
stain darker at that spot. Avoid crushing
the gel with fingers or dish when decant-
ing solutions or transferring gel to a new
solution.
Section 8
Ordering Information
Catalog
Number Product
Description
161-0443 Silver Stain Kit, includes 1 bottle oxidizer concentrate, 1 bottle silver
reagent concentrate, and 4 bottles developer. Enough to stain approxi-
mately 24 16 cm slab gels.
161-0444 Oxidizer Concentrate , 480 ml
161-0445 Silver Reagent Concentrate , 480 ml
161-0447 Silver Stain Developer , 4 x 115 g
9
Troubleshooting Guide (continued)
Problem Solution
Temperature too low. Make sure temperature
of all silver stain reagents is at least 23 ˚C.
Mirroring can be caused by the developer
precipitate sticking to the gel surface. Be sure
to decant first development solution as soon
as the precipitate appears (see protocol).
Dark uniform background, usually Oxidizer is not completely removed.
Increase
yellow.
wash steps 5, 6, and 7 as described above,
removing all traces of yellow, before
going on to silver reagent step.
Mottled background, usually Contaminants in water. Check that the
brown or green, with poor sensitivity. conductivity of the water is less than 1
µmho. See "Notes on Silver Staining."
Incomplete removal of gel buffer compo-
nents. Increase times of steps 1, 2, 3 to
insure complete removal.
Slow or no development. Rate of development is highly temperature
dependent. Developer solution can be
heated to 50 ˚C to speed development.
Developer solution is too old
(paraformaldehyde has evaporated). Shelf
life of solution is 1 month at 23-25 ˚C.
Settling and separation of the components
of the developer powder will occur. When
preparing solutions of less than 3.6 liters,
mix the contents thoroughly before
aliquoting.
Gel continues to develop or becomes Increase stop time (step 13) and change stop
darker upon drying onto filter paper. solution 2 or 3 times to remove all devel-
oper. Dry between two pieces of clear cel-
lophane or photograph before drying onto
filter paper.
8
LIT34B 6/17/98 2:04 PM Page 8