3/4/96

Good morning,
I've recently been trying to do immunolocalization at the EM level with affinity-purified Ab. My problem is that the purified Ab's are in an ammonium thiocyanate solution that chews up both the Ni grids and the sections (LR White). I know it's the solution because the polyclonal serum immunos are fine. Does anyone with experience purifying Ab's and using them for TEM immuno care to chime in with some pertinent advice? I'd appreciate hearing any and all suggestions. Many thanks!

Dwight Beebe
beebed@ere.umontreal.ca

Dwight --
Try dialyzing the affi-pure Ab into Tris-Buffered Saline. I use 10mM Tris, 500mM NaCl, pH 7.2 for use on LRGold sections. Avoid phosphate as it causes lead stain pption.

Greg Martin
gmartin@welchlink.welch.jhu.edu

To Dwight Beebe:
Antisera can be easily separated from undesirable salts by dialysis...this is by far the simplest method. Obtain a short length of dialysis tubing, soak it in water or buffer which will allow it to be opened up into a tube. Tie off one end, fill with your antibody solution, then tie off the other end. Immerse it into whatever buffer you want to antibody to end up in (I suggest either TRIS buffered or phosphate buffered saline. Use 1000X as much buffer as antiserum, ie dialyse 1 cc of antibody against 1 liter of water for 24 hours, then change the buffer and continue for another 24 hours. I recommend the last buffer change to contain 0.001 mM sodium azide to retard microbial growth.

This will hopefully solve your problem.

W. L. Steffens, Ph.D
STEFFENS.B@CALC.VET.UGA.EDU

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