After adding RIPA lysis buffer (Cat.No.: HY-K1001, bought from MCE) to cells, white floccules appear. Is this normal?
When I used RIPA lysis buffer to treat adherent/suspended cells, I observed white floccules in the system:
Phenomenon:
After adding lysis buffer (according to the recommended ratio) and lysing on ice, white floccules can be seen with the naked eye. After centrifugation, the precipitate is insoluble in the supernatant. May I ask if the following situations may cause this phenomenon?
Question:
Does the lysis buffer cause salt or detergent components to precipitate due to low temperature storage (such as long-term storage at 4℃)? Does it need to be incubated at 37℃ for re-dissolution before use?
Does the protein content or extracellular matrix of the target cells (such as primary cells/tumor cell lines) cause a large amount of cell debris/protein complex precipitation?
[Troubleshooting Questions are selected from MCE customer consultation emails.]
Phenomenon analysis and treatment suggestions
1. Determination of the normality of the phenomenon
The appearance of white flocs in the lysis system is a common experimental phenomenon, mainly caused by biomacromolecule aggregates produced during cell lysis, and does not affect the conventional protein extraction process (non-extreme abnormal conditions).
2. Possible causes and material composition
Cell membrane fragments and lipid aggregates: Cell membrane components (such as phospholipid bilayers) released after cell rupture react with detergents (SDS/Triton X-100) to form micelle structures, which are prone to physical aggregation at low temperatures;
Incompletely dissolved protein complexes: High-abundance proteins (such as cytoskeletal proteins) or hydrophobic proteins are insufficiently soluble in the lysis solution, forming flocculent precipitation visible to the naked eye;
Genomic DNA-protein complexes: Genomic DNA released during the lysis process can bind to histones and the like through electrostatic interactions to form a network precipitation structure.
3. Experimental optimization operation suggestions
- Cell quantity control: Optimize the cell seeding density through preliminary experiments (recommend to reduce 20%-50%) to avoid protein overload of the lysis system due to excessive cell concentration;
- Centrifugation treatment scheme: Applicable to the case where proteins that are particularly tightly bound to genomic DNA are not detected. Use low temperature (4℃) high-speed centrifugation (12,000-15,000×g, 10-15 min), and take the supernatant for subsequent experiments (applicable to non-histone related detection scenarios).
Operation points:
This phenomenon does not affect the extraction efficiency of soluble proteins. The supernatant after centrifugation can be directly used for conventional experiments such as Western blot and IP, but the subsequent processing steps need to be adjusted according to the characteristics of the target protein.
The answer to this question comes from MCE Technical Support.
How to contact us?
- Tel: 609-228-6898
- Fax: 609-228-5909
- E-mail: [email protected]
- Technical Support: [email protected]
- Address: 1 Deer Park Dr, Suite F, Monmouth Junction, NJ 08852, USA
- Badges
- Science method