Dear Lucian Miles

theorerically short term vortexing do not have to affect the folding of any protein or antibody.

the only concerns may be related to indirect effect of long term vortexing:

- Oxidative stress due to fast shaking and higher aeration that could be avoide with long gentle agitatiion ( e.g with a magnetic stirrer) However if the vortexing will really negativelly affect depends a lot protein, how much aminoacids that may be susceptible to oxidation (e.g Methionine or Tryptophans) are exposed in the surface and if those aminoacid are essential or not for your protein structure and function or not.

- Increase in temperature that of course if the sample reach temperature higher than 37°C may be deleterious.

However i think that those are extreme effect that comiing only if you are vortexing the sample for long time (minutes and not seconds)

best

Manuele

For cytokine recombinant proteins, their biological activity is highly dependent on the correct spatial structure, so the dissolution operation should avoid destroying the protein conformation.

The specific suggestions are as follows:

1. Use a pipette tip to gently blow or turn it upside down to mix, and avoid severe mechanical force to damage the protein structure.

2. If the dissolution is not sufficient, the reconstituted protein solution can be placed on a horizontal shaker (low speed) for oscillation, or left to stand at 4°C for more than 2 hours, and the dissolution can be promoted by gentle stirring or prolonged incubation time.

This operation plan is designed to maintain the natural conformation and biological activity of the protein to the greatest extent, and is suitable for cytokine recombinant proteins that are sensitive to spatial structure.

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Sorry , not my exact field of specialization?

1. Can vortexing cause protein structure destruction or aggregation?

Yes, vortexing can damage recombinant proteins. The intense shear forces introduced during vortexing can disrupt the protein’s tertiary or quaternary structure, leading to partial or full denaturation. This process may also introduce air bubbles, increasing the risk of oxidation and promoting protein aggregation. As a result, the protein may lose its biological activity or form insoluble clumps or non-functional polymers. This is especially problematic for sensitive proteins such as enzymes, membrane proteins, or antibodies, which rely heavily on correct folding for their function.

2. Do different types of recombinant proteins have different tolerances to vortexing?

Yes, different types of recombinant proteins vary in their sensitivity to vortexing. Intracellular proteins—particularly those expressed in bacterial systems—are more prone to misfolding and often form inclusion bodies, making them more sensitive to mechanical stress like vortexing. Secreted proteins, on the other hand, typically undergo more thorough folding and quality control during synthesis (e.g., in the endoplasmic reticulum and Golgi apparatus), so they may tolerate mild agitation slightly better. However, no recombinant protein is immune to damage from vortexing, so caution is always advised.

3. If vortexing is not suitable, what alternative methods are recommended for dissolving proteins?

There are several gentler and more protein-friendly methods to dissolve recombinant proteins:

Gentle inversion: Slowly turning the tube end-over-end at room temperature or 4°C is often sufficient, especially for lyophilized proteins.

Slow stirring: Using a magnetic stir bar with slow rotation helps dissolve proteins gradually without damaging their structure. This method works well with proteins stored in stabilizing solutions.

Bath sonication: Placing the tube in a low-power ultrasonic water bath for a few seconds can help break up soft aggregates. Avoid using probe sonication unless it has been optimized for your protein.

Mild warming: Incubating the protein solution at room temperature or up to 37°C for a short time can improve solubility. However, this should be done cautiously to avoid thermal degradation.

Buffer optimization: Adding stabilizing agents such as glycerol (5–20%), arginine or glutamate (100–400 mM), or non-denaturing detergents like Tween-20 (0.01–0.1%) can help maintain solubility. For very stubborn proteins, low concentrations of urea (1–2 M) may be used, provided there is a suitable refolding protocol.

4. What if the protein takes too long to dissolve?

If protein dissolution is unusually slow, start by ensuring that the buffer pH, ionic strength, and additives are appropriate for the protein’s properties. Try using small buffer volumes first and dissolve the protein in stages. Always add buffer gently along the wall of the tube to avoid foaming. Use gentle rotation at 4°C for prolonged periods—overnight if needed—rather than resorting to harsh methods.

Avoid repeated freeze-thaw cycles, as they often lead to aggregation. Instead, prepare small working aliquots of the protein and store them appropriately. If you notice persistent clumping, it might be necessary to briefly centrifuge the sample at low speed or filter it through a 0.22 µm syringe filter.

Summary and Expert Advice

To protect recombinant protein activity, avoid vortexing unless absolutely necessary. Instead, rely on gentle methods like inversion, slow stirring, and mild temperature increases. If the protein remains insoluble, review the buffer formulation or consider modifying the expression system or purification tags. Ultimately, patience and protein-specific optimization are key. If you're working with a particular protein (e.g., cytokine, enzyme, or antibody), more tailored advice can be provided.

Hezam Dhaifallah Thanks & Thank you for providing such detailed answers to my questions. It helps me a lot!

Zainab Kadhim Hasan It's OK, thanks your notion for this question.