Aflatoxin M1 Immunoaffinity Column

Additional Information

Enrichment Step Immunoaffinity Chromatography (IAC)

50ml skimmed milk are diluted with 5ml 10X PBS (= 10fold concentrated 50mM PBS buffer) and then applied in a reservoir on top of the B-TeZ IAC Aflatoxin M1 3ml column. Rate of flow through the affinity gel is 1 to 3 ml/min.
Caution! Be aware that no big air bubbles are neither in the gel nor between gel and luer lock outlet of column which prevent a permanent flow or necessary exchange of matter.
Depending on application and on expected contents, larger or smaller extract aliquots can be applied. In such cases the sample calculation (see below) must be adapted. If samples are to be prepared simultaneously, manifold of J.T. Baker for 12 samples has proven of value.

Wash

After whole sample has passed through the gel the latter is washed with 5ml of 50mM PBS. Remaining liquids in the gel are removed by applying either pressure from top of the column or underpressure from bottom.

Elution

Sample reservoir on top of the B-TeZ IAC Aflatoxin M1 3ml column is removed and a appropriate vial is placed below the affinity column. The bound toxins are eluted by using a total of 1.5ml of methanol as elution solvent. The elution process is performed in two steps to ensure complete release of analytes. First, a volume of 0.5ml elution solvent is applied. After that volume has passed through column half a minute is waited before the second portion of 1ml of elutions solvent is eluted through the column. Remaining solvent solutions should be eluted by application of slight under or overpressure. All methanolic fractions are unified to give the column eluate. The column elute may be injected into the HPLC directly or in case concentrations are low it may be concentrated by evaporation (e.g. using VLM evaporater at 50°C under a permanent stream of nitrogen).
Caution: If you follow concentration by evaporation, add 100 l of acetic acid/water (50/50 v/v) as keeper. To avoid loss of analytes, stop concentration at a small volume of residue, e.g. 50 to 100 l.
The residue then is redissolved in HPLC solvent, e.g. in 400 l in example calculation see below, and an aliquot is finally injected into the system.