Affinity Purification of Monospecific Antibodies on Western Blotted Antigens (specific example for purification of anti-IC140 from Chlamydomonas inner arm I1. But applicable to others)
Solutions:
TBS (Bio-Rad formula)2) Blot overnight, 0.5Amps = 25-30V.
3) Stain blots with Ponceau S ~ 1Hour.
4) Carefully cut out relevant bands from the blot (140, 97kD, HCs, and other bands, and a non-staining region above 140). The blot strips can be wrapped in saran wrap and stored at -80 degrees C.
5) Place blot strip in 15ml orange-top tube, wash 2X in 9ml T-TBS, 10 minutes each.
6) Block in 3% gelatin in TBS, at least 1 hour. (3g/100, .3g/10)
7) Wash once in T-TBS.
8) Add 250m l of Eu-7 sera per 10ml of T-TBS with 1% BSA (=Ab solution), incubate strip in this at 4 degrees C, rocking for 5-4 hours or overnight. (4-5 hours is much better) (1g/100, .1g/10)
NOTE - Perform all subsequent operations at 4 degrees C.
9) Remove (pour off) Ab solution, save this (for subsequent purification of anti-97).
10) Wash 2X, 10 minutes with T-TBS, pull off all with pipette.
11) Wash 2X, 20 minutes with TEN, remove thoroughly.
12) Using clean gloves, fold blot strip into an accordion about 1cm in length, place on a clean petri dish and cut into thin slices with an ethanol cleaned razor blade.
13) Place these blot chips into syringe apparatus constructed of two 5ml syringes connected by about 2cm of tygon tubing (3/32 ID, 5/32 OD, 1/32 wall). It's best to put a little (1-2ml) of TEN in the syringe to help wash the chips off the forceps and prevent them from drying out too much. Then pull off TEN with a pulled-out Pasteur pipet.
14) After all chips are in the syringe and the wash solution is totally pulled off, add 3ml of the Glycine solution and push back and forth gently. To do this, apply slight back pressure to the syringe apparatus. Don't let this or anything build up much pressure, otherwise the tubing will blow off the syringe. The slight back pressure minimizes bubble formation (due to the detergent) which can denature the proteins.
15) After 4-4.5 minutes, remove the Glycine solution with a fresh pulled out Pasteur pipet and put into 0.3ml of 1M Tris. (Any longer in pH 2.8 and you risk denaturing the Abs)
16) Add 6ml of TBS and 2 drops of 1N NaOH.
17) pH to 7.2-7.4
18) Add 0.02% Na-Azide (=1/100 of 2% stock)
19) Add BSA to 1%, store at 4 degrees C for a couple of weeks or freeze in IN2 and store at -80 degrees C.
20) Wash the blot chips with T-TBS if they are to be reused.
NOTES - When using blot chips, always use a pulled-out pasteur pipet to remove all solutions and prevent loss of them or contamination with the tiny blot chips.
The blot chips can be used at least twice more. Wash 2 more times with T-TBS, 10 minutes each before adding more Ab solution. Perform incubations and elutions as before.
Except, blot chips are most easily transferred by transferring them as suspension in whatever buffer they are in. All incubations are best carried out in the 15ml orange, screw-top tubes. To transfer to syringe, leave 3-4ml in the incubation/wash tube and carefully pour into the syringe.
After each wash or incubation step using chips, just stand the tube up off rocker to the chips settle to the bottom. Then the solution can be poured off and the last 0.5ml or so can be removed with a pulled out pipet.