How long can the protein without loading be stored at -80°C? Will there be any impact on BCA after thawing?
Protein samples without loading buffer can be stably stored at –80°C for about 1 month, and should be used promptly if no protease inhibitor is added. Generally, performing BCA protein quantification after thawing will not pose significant issues, as long as repeated freeze-thaw cycles are avoided and operations are conducted promptly; however, it should be noted that freeze-thaw may lead to partial protein degradation or aggregation, which might slightly affect quantification results.
1. How long can it be safely stored?
1. Protein samples without loading buffer, if in cell lysate or tissue homogenate (such as RIPA lysis), can generally be stably stored at –80°C for 1 to 3 months.
2. However, the shorter the duration, the better, and it is recommended to use within 1 month, as proteins may gradually degrade or denature, especially after protease inhibitors become inactive.
3. If no protease inhibitor (such as PMSF, cocktail, etc.) was added before storage, it is recommended to use the samples promptly, ideally not exceeding 1–2 weeks.
2. Will thawing and subsequent BCA protein quantification be affected?
There is some impact, but usually not significant, provided that:
1. Samples remain frozen and are only thawed once;
2. The lysis buffer contains an appropriate amount of protease inhibitor;
3. BCA quantification is conducted immediately after thawing, avoiding prolonged exposure at room temperature or 4°C.
Note:
1. Freeze-thaw processes may lead to partial protein denaturation and aggregation, which might slightly affect BCA results (sometimes underestimating concentration);
2. BCA is sensitive to reducing agents, detergents, etc., so verify compatibility of the buffer system in the lysate with BCA before conducting the assay after thawing.
3. Recommended operations
1. Immediately aliquot and add loading buffer to thawed samples, heat-denature, then perform WB;
2. If performing BCA, do so promptly to prevent repeated freeze-thaw cycles;
3. If protein concentration is critical for the experiment, consider conducting parallel quantifications on multiple samples for comparison.
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