Can you fix annexin V staining?
In my experience, it is possible to fix annexin V stained cells and see great results on the flow. Stain cells with annexin V (in calcium-rich binding buffer). Wash cells in binding buffer to remove unbound AxV. Fix in your choice of fixative (diluted in binding buffer).
Can you fix Annexin V?
How do you increase protein purity?
Increasing the imidazole concentration during binding and washing should improve protein purity although you may also get some loses of your protein. We make use of ReSyn Ni-NTA beads (see link below) and use 80mM imidazole during binding /washing.
Can annexin be fixed?
How do you know if a protein is pure?
Generally, we can check the purity by quantification methods like UV-Vis, Bradford and Activity Assays. Meanwhile, electrophoresis is widely used by biochemists and can provide a general picture of both the size of your target protein whether other protein-based impurities present.
How does pH affect protein purification?
Why is pH important in affinity purification? The pH of a solution determines the physical states of the proteins (charge, etc) contained within based on the pKa values of their amino acids. Different proteins have different ranges in which they are stable or will bind to other proteins.
What is the annexin V protocol used for?
The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells 3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5.
Why are annexin V reagents used in apoptosis?
Because of this affinity, fluorescently labeled annexin V reagents are commonly used in apoptosis research. Annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine, an indicator of intermediate stages of apoptosis.
What is annexin V APC conjugate?
Contains 1 vial of annexin V, APC conjugate. Annexin V has a high affinity for phosphatidylserine (PS), which becomes exposed on the outer leaflet of cells undergoing apoptosis. Because of this affinity, fluorescently labeled annexin V reagents are commonly used in apoptosis research.
How do you store annexin V Alexa Fluor 488 conjugate?
Store in refrigerator (2–8°C) and protect from light. Unlike other Annexin V kits that have lower protein concentrations or purification levels, the Annexin V Alexa Fluor 488 conjugate is optimized for flow cytometry to provide the largest separation between apoptotic and live cells.