Protein & Antibody

Optimize elution conditions by gradually lowering the pH from a high value; neutralize the purified sample promptly, or even neutralize while purifying; prepare and store the sample at low temperatures during the purification process; add protein protectants such as glycerol and Tween; when antibody concentration is high, dilute in time to avoid the risk of aggregation due to high concentration.

The verification can be done from the following three aspects:

First, at the molecular sequence level, after constructing the expression vector, the expression plasmid is sequenced for confirmation. Sequencing files can be used for verification.

Second, during the expression process, a comparison is made between induced and uninduced lysis buffers. Differences in protein bands can be detected by SDS-PAGE. The difference can be observed at the theoretical size of the target protein.

Lastly, Western blot (WB) validation can be performed using tag antibodies or antibodies specific to the target protein.

If protein verification is needed, protein coverage analysis can be conducted by comparing the theoretical sequence with the actual coverage ratio. However, the purity of the target protein must be greater than 90%. If the purity is lower, the coverage ratio may be affected. For lower purity samples, gel slicing can be performed for further analysis.

The advantage of eukaryotic expression systems over prokaryotic systems is that the protein activity is generally better. In theory, most proteins expressed in eukaryotic systems are active. However, the stability of different samples may vary, so the actual experimental results should be used as the basis for determining protein stability.

Advantages of CHO Cells:

• Can grow stably in chemically defined, serum-free suspension cultures.

• Well-characterized genome and generally safe with respect to human pathogenic viruses.

• Capable of human-like post-translational modifications (e.g., glycosylation).

• Amenable to genetic modification and stable cell line generation.

Advantages of HEK293 Cells:

• Faster growth rate compared to many other mammalian lines.

• High cell density and very high transfection efficiency.

• Produce post-translational modifications that closely resemble human protein structures.