How long can WB samples without loading buffer be stored at -80°C? Is it true that membrane proteins cannot be boiled? Do we need to perform BCA every time?

WB samples without loading buffer stored at -80°C can be viable for short-term (1-2 weeks), but long-term storage is not recommended. Boiling is generally not advised for membrane proteins; instead, use low-temperature incubation or gentle lysis methods. It is recommended to perform BCA protein quantification each time unless experimental conditions are extremely stable. Below is a detailed explanation:

 

1. How long can WB (Western Blot) samples without loading buffer be stored at -80°C?

1. Short-term (few days to weeks): If the samples are cell lysates or protein extracts, storing them directly at -80°C without loading buffer usually preserves them for 1-2 weeks without noticeable degradation.

2. Long-term (months or more): Proteins may degrade or denature, especially with repeated freeze-thaw cycles. If planning for long-term storage, it is recommended:

(1) Add SDS loading buffer and boil as soon as possible (if allowed by the experimental design).

(2) Add protease inhibitors to reduce degradation.

(3) Avoid repeated freeze-thaw cycles by aliquoting samples into smaller portions.

 

2. Is it true that membrane proteins should not be boiled?

1. Membrane proteins are generally not recommended to be boiled because:

(1) Boiling (95°C, 5 min) can cause some membrane proteins to aggregate, denature, or precipitate, affecting electrophoretic separation and transfer efficiency.

(2) Many transmembrane domains of membrane proteins are unstable at high temperatures, which may affect antibody recognition.

2. Solutions

(1) Lower denaturation temperature: Generally, incubate at 37°C~55°C for 10-20 min instead of high-temperature denaturation at 95°C.

(2) Use mild denaturation methods: Such as buffers without DTT, β-mercaptoethanol, or SDS.

(3) Select appropriate lysis methods: Use mild detergents like NP-40 or Triton X-100 for extraction instead of SDS lysis.

 

3. Is it necessary to perform BCA protein quantification every time for WB?

1. Theoretically, yes, it is recommended to perform BCA quantification each time.

2. Exceptions:

(1) If using the same cell line, same lysis method, and identical experimental conditions with stable results from previous experiments, BCA may not be necessary, and adjustments can be made based on WB band intensity.

(2) If the protein sample has been stored for a long time or its concentration might have changed (due to freeze-thaw or different extraction batches), it is best to redo BCA to ensure consistent loading amounts.

 

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