S I L V E R S T A I N I N G
(For a PAIR of 1.5 mm thick gels)
GLASS DISTILLED WATER SHOULD BE USED

Reagent Milliliters Duration
1. Fixative-I (200 ml H₂O + 160 ml EtOH + 40 ml HOAc) 400 1 to many hours
2. Fixative-II (680 ml H₂O + 80 ml EtOH + 40 ml HOAc) Use half! 400 30 min
3. Fixative-II Use other half! 400 30 min
4. Per-Iodic Acid Oxidizer* (can be reused for about a week) 400 10 min
5. H₂O (a quick, little slosh) approx 100 a few seconds
6. H₂O (These waters are flushing away anions that will interact with silver in step 9) 400 10 min
7. H₂O 400 10 min
8. H₂O 400 10 min
(At this point, start making the slow dissolving developer of step #12; magnetic stirrer)
9. Silver Rgt. (20 ml BioRad Silver [refrigerated] + 180 ml H₂O)
DO THIS IN THE DARK. Cover with a box. 200 30 min
10. H₂O (a quick, little slosh) approx 100 a few seconds
11. H₂O 400 2 min
12. Developer (11 g BioRad Developer [refrigerated] + 600 ml H₂O)
(Don't get any undissolved specks of developer onto gels!) 200 2 min
13. Developer 400 until dark enough
14. Stop Solution (400 ml H₂O + about 20 ml HOAc) 400 5-10 min
15. Drying Solution (200 ml EtOH + 200 ml H₂O) 400 overnight
16. Cellophane dryers (not Saran Wrap) ≥ 24 hrs

Reagent	Milliliters	Duration
1. Fixative-I (200 ml H₂O + 160 ml EtOH + 40 ml HOAc)	400	1 to many hours
2. Fixative-II (680 ml H₂O + 80 ml EtOH + 40 ml HOAc) Use half!	400	30 min
3. Fixative-II Use other half!	400	30 min
4. Per-Iodic Acid Oxidizer* (can be reused for about a week)	400	10 min
5. H₂O (a quick, little slosh)	approx 100	a few seconds
6. H₂O (These waters are flushing away anions that will interact with silver in step 9)	400	10 min
7. H₂O	400	10 min
8. H₂O	400	10 min
(At this point, start making the slow dissolving developer of step #12; magnetic stirrer)
9. Silver Rgt. (20 ml BioRad Silver [refrigerated] + 180 ml H₂O) DO THIS IN THE DARK. Cover with a box.	200	30 min
10. H₂O (a quick, little slosh)	approx 100	a few seconds
11. H₂O	400	2 min
12. Developer (11 g BioRad Developer [refrigerated] + 600 ml H₂O) (Don't get any undissolved specks of developer onto gels!)	200	2 min
13. Developer	400	until dark enough
14. Stop Solution (400 ml H₂O + about 20 ml HOAc)	400	5-10 min
15. Drying Solution (200 ml EtOH + 200 ml H₂O)	400	overnight
16. Cellophane dryers (not Saran Wrap)		≥ 24 hrs

* "Per-Iodic" Acid: 40% EtOH, 5% HOAc, 0.7% H₆IO₅ (220 ml H₂O + 160 ml EtOH + 20 ml HOAc + 2.8 gm per-iodic acid).

(This solution can be re-used about 8 times or for 1 week, whichever comes first. It should not smell like ethyl acetate [fingernail polish remover])

Tsai, C-M., and C. E. Frasch. 1982. A Sensitive Silver Stain for Detecting Lipopolysaccharides in Polyacrylamide Gels. Anal. Biochem. 119: 115-119. (THE ORIGIN OF SILVER STAIN. One of Dr. V's students worked with Tsai at N.I.H. in Washington DC.)

Kropinski, A. M., D. Berry, and E. P. Greenberg. 1986. The Basis of Silver Staining of Bacterial Lipopolysaccharides in Polyacrylamide Gels. Cur. Microbiol. 13: 29-31. (About the theory of the silver stain.)

Hitchcock, P. J., and T. M. Brown. 1983. Morphological Heterogeneity among Salmonella Lipopolysaccharide Chemotypes in Silver-Stained Polyacrylamide Gels. J. Bacteriol. 154: 269-277. (Use of per-iodic acid for the visualization of LPS in SDS-Page.)