Hi,

Answers to your questions:

1. No, but yes. The induction will work at any volume, but if you have low expression, you might not get a detectable amount of protein. Further, I find that proteins are best expressed in a baffled Erlenmeyer flask. If you're using a 15 mL tube to make a 2 mL culture, you might find that the expression is lower than in a larger volume in a baffled flask.

2. Sonication is not necessary. You can lyse the cells with a cell lysis buffer or with a French press and get good results. Freezing and thawing could work, but I do not have much experience with that method.

3. Your IPTG might be too old. The best way to check it is to use it on a positive control strain.

Other things to consider:

A. Optimizing your codons for E. coli or using an expression strain that uses rare codons (I like the Rosetta strain for this. https://www.emdmillipore.com/US/en/product/RosettaDE3-Competent-Cells-Novagen,EMD_BIO-70954)

B. Use a longer induction time at a lower temperature. Overnight induction at 18 degrees is my typical protocol.

C. Do a rough purification before checking for protein production. Sometimes the target protein is close in size to an abundant native protein and it can be difficult to distinguish this from a whole cell lysate. Prepare an uninduced control alongside of your sample and purify both before running on a gel.

Thank you Ryan Wheeler for your suggestion. This is really helpful.