Recombinant Proteins Support—Troubleshooting

Gibco recombinant proteins are frequently formulated without carrier proteins or additives (e.g., BSA, HSA, sucrose, etc.) and no Gibco PeproTech recombinant protein contains a carrier protein. As a result, during lyophilization, the protein product may be deposited on the vial as a thin, and sometimes invisible, film instead of a pellet. The size of the pellet, if any, is not directly related to the quantity of the recombinant protein in the vial. Our quality control procedures assure that each vial contains the correct amount of product.

To ensure complete recovery of protein product, before opening a vial of lyophilized recombinant protein, we recommend centrifuging it in a microcentrifuge for 20-30 seconds to drive any protein that may be lodged in the cap or on the side to the bottom of the vial. After reconstitution, you can confirm the presence of product protein by running a small amount 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. 

The solubility issue might be due to the improper handling, or use of a solvent other than the one we recommended. We recommend that you warm the lyophilized powder to room temperature before you open the vial, and that you solubilize the protein in the buffer solution recommended in the manual (some proteins are more soluble in low pH buffer). Do not reconstitute at a protein concentration >1 mg/mL. Do not vortex or mix protein solutions vigorously. Allowing the reconstituted protein to incubate overnight at 4°C may help resolve any solubility issues.

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 <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.

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.