Dear Sumit

is your protein soluble?

did you incubate the full lisate or only the soluble fraction after removal or the pellet with centrifugation?

Manuele

Hi Manuel,

it is a soluble protein and I used the soluble fraction.

To verify your statement, your protein does not degrade in the feed, neither does it degrade in the lysate at low temperature, correct? If so, I wonder what buffer you used to elute your protein from the beads, and how much it is different from your feed conditions.

Hi Jinxiang,

Following IP, I just add SDS sample buffer to the beads and boil it. Then following separation of the magnetic beads (just by placing against the magnet), collect the sup. It is the same buffer that I used for the feed. Also, In my feed I see three bands (67, 54 and 47KDa) while in the IP fraction I see 50, 35 and 27Kda.

Sumit, I am not a big fan of boiling my proteins. Many proteins are pretty fragile at high temperature. You may try to boil your feed to verify it. If you care about the integrity or functionality of your protein, I suggest you use HA tag peptide to elute the protein. Otherwise, you may explore elution using 0.1M glycine, pH 2.0 or 50mM NaOH, if your protein is not sensitive to low or high pH conditions. I hope it helps.

Hi,

Is it the Dynabeads Protein G that are used as solid support - non covalent binding? If low yield of target have different anti-HA been tested?

Ab & target binding

Ab binding to the Dynabeads is usually very fast due to rapid binding kinetic. We have performed several experiments to demonstrating that increasing the time for Bead:Ab incubation is not required. Extending the incubation time to 1h or even overnight will not result in much more Ab binding. The binding efficiency will be dependent on the bead type and the Ab subclass, e.g. IgG1 will bind efficiently to Prot G but to less extent to Prot A beads. When incubating your Dynabeads coated with Ab with the lysate containing target one have to optimize that as this will depend on your primary Abs affinity for the Ag. In our in house experiment targeting CD9 very nice signal on western blotting after only 10 min incubation with target is obtained (both manually & automated).

Elution

Typically mild elution methods include change of pH and change of ionic strength Protocols may need to be optimized dependent on the antibodies affinity towards the target. The coated Dynabeads with antibody may be reused several times using mild elution methods like low pH or high salt concentration. The success of elution depends of course on affinity of immobilized antibody and the captured target. In addition, the reusability of the immobilized antibody using low pH also depends on sensitivity of the immobilized antibody toward this type of treatment. If possible using high salt concentration as elution method assures several rounds of reusing beads but using low pH, the yield will normally decrease after couple of rounds of reusing

The elution efficiency depends on the volume. Increased volume will result in higher elution efficiency. By increasing the elution volume from 20 µL to 100 µL will increase elution efficiency by 20 %. However, using 20 µL is related to the well size of the gels. This needs to be optimized for each immunoprecipitation.

If the bead-target complex is boiled directly in SDS the prim Abs will be eluted off together with the target (we switched to LDS for 10 min at 70C and found that that worked better for our target proteins). To avoid detection of the heavy and light chain from the capture Abs when doing western we have used the TrueBlot secondary Abs conjugated with HRP with success.

kind regards

ketil