Recombinant Proteins Support—Troubleshooting

If you carried out a test as described in our product insert and did not see any response, this could be due to several possibilities listed below:

• Protein was not reconstituted according to the instructions.
• The reconstituted protein is too old or protein might have precipitated. We recommend using the reconstituted protein within 3–6 months after reconstitution.
• Carrier protein was not added for proteins reconstituted at a concentration is <0.1 mg/mL. Working solutions at <0.1 mg/mL should be used immediately; we do not recommend long-term storage of solutions at this concentration.
• The protein solution was exposed to multiple freeze/thaw cycles or was exposed to high temperature.
• Proteins were handled in the wrong types of vessels (some proteins are very sticky to certain plastics).

Assay time is critical. Each assay needs to beoptimized and performed at the peak response time. Different cells may respond differently to a growth factor or cytokine. We suggest repeating our QC assay using same indicator cells as suggested in the manual to see if you can obtain a similar response. In addition, serum may be masking the response. Serum starvation might be needed for certain types of assays.

Our recombinant protein is shipped as lyophilized powder. In most cases, a pellet is visible in the tube or sometimes the pellet has become dislodged and is stuck in the cap or on the side of the vial. A brief centrifugation should bring the pellet to the bottom. However, not all lyophilized proteins are visible with naked eye. In most cases, the visible white pellet is the salt that was present in the original buffer solution, and the size of the pellet may not be directly related to the quantity of the recombinant protein in the vial. If the recombinant protein was in salt-free solvent before lyophilization, the pellet may not be easily visible (it often has a transparent appearance). You can confirm the presence of protein by running a small amount of reconstituted solution on SDS PAGE. In general, a protein band with expected size should be visible with as little as 10 ng of protein loaded on an acrylamide gel.

If the assay does not give a sigmoidal response curve for a given cytokine or chemokine, then no ED50 information will be given. For example, the response curves for many chemotaxis assays are bell shaped rather than sigmoidal (in this case, there will be two concentration points that give 50% of maximum response; one is lower than the concentration induced maximum response, the other at higher concentration). For some of the cytokines and chemokines if the biological activity is determined by chemotaxis assay, we often provide a concentration which gives a significant response.