Veronica Baldoneschi

Below is my typical elution protocol that removes high background (used this protocol for Co-IP studies that lead to mass spec).

1. Wash Beads in wash Buffer ( 50 mM Hepes and 150 mM NaCl, pH 7.4) to remove storage buffer

2. Add 400 uL of magnetic beads per 1000 uL aliquots

3. Add 1 mL of (50 mM Hepes, pH 7.4, 150 mM NaCl, 1.0 mM EDTA, 1% Trition X-100) with 10 uL of MG132 and 50 uL of protease inhibitor cocktail per eppendorf tube

4. Incubate for 2hrs gently rotating @4 degrees

5. Place eppendorf tubes in magnetic separator and quickly removed supernatant and safe it for unbound analysis

6. Add 1 mL of Low Salt Buffer (20 mM Hepes, 150 mM NaCl) per eppendorf tube

7. Gently rotate @ 4 degrees for 5 minutes

8. Place eppendorf tubes in magnetic separator and quickly remove supernatant

9. Add 1 mL of High salt buffer (20 mM Hepes, 500 mM NaCl) per eppendorf tube

10. Gently rotate @ 4 degrees for 5 minutes

11. Place eppendorf tubes in magnetic separator and quickly remove supernatant

12. Add 1 mL of LiCl salt buffer (20 mM Hepes, 250mM LiCl) per eppendorf tube

13. Gently rotate @ 4 degrees for 5 minutes

14. Place eppendorf tubes in magnetic separator and quickly remove supernatant

15. Add 100 uL 25 mM glycine-HCl buffer pH 2.5 per eppendorf tube to elute off beads

16. Add 600 uL of 50 mM Hepes, pH 7.4, 150 mM NaCl to each eppendorf tube

17. Gently rotate overnight @ 4 degrees

18. The next day Place eppendorf tubes in magnetic separator and quickly remove supernatant and place eluted fraction in fresh eppendorf tubes

19. Add equal volumes of -20 Acetone to each eppendorf aliquot and incubate on ice for 40 minutes

20. Leave in -20 overnight

21. The next day spin tubes at 14000 rpm for 30 minutes at 4 degrees

22. Carefully remove Acetone, letting the pellet air dry

23. Resuspend with 10 uL of water and 10 uL of 2X Laemmli buffer

24. Load on gradient acrylamide electrophoresis gel

Hi there,

What are the washing steps you use prior to elution?

There are two general strategies for reducing non-specific binding. Consider using either or both strategy, and try various combinations to see what gives you the best result. The best solution mostly depends on your antibody-target interaction.

One strategy is blocking non-specific binding in the first place. First, include 0.1% Tween in all of your buffers. Second, always pre-incubate your beads with a blocking buffer before you put them into your human serum. You can use a commercial ELISA blocker, like Pierce/Thermo SuperBlock, or try 1% non-fat milk or purified BSA in PBS. Third, pre-dilute your serum in a buffer that allows binding of your target but inhibits non-specific binding. Try up to 1M NaCl, 1M glucose, and 1% Triton in different combinations.

The others strategy is differential elution. First wash off the non-target materials, while leaving your target bound to the beads. Try monocationic salts as Krueger described, detergents, and the blocker ingredients listed above. Second, denature to elute your target in a different fraction from the undesired material. You have tried the standard elution at pH 2 (followed by facile neutralization). You can also try intermediate pH. See what comes off at pH 3 or pH 4, for example. Also try chaotrope elution using guanidine HCl at various concentrations up to 8M (followed by dialysis or buffer exchange on a column).

Two more thoughts:

The avidin-biotin reaction is generally susceptible to background binding. I have seen much lower background using anti-biotin antibodies to pull down biotin labeled targets, instead of streptavidin.

Your target may naturally bind to non-target materials and co-precipitate them. Chaotropes are useful for controlling this effect.

Dear Dominique Liger I wash two times with TBS pH 7.2 + 0,1% tween