Gelatin-agarose gel 4FF: separation reagent, chromatographic medium.

Gelatin-agarose gel 4FF is a kind of affinity chromatography filler which is coupled to agarose gel 4FF matrix by cyanogen bromide method. Gelatin is a polymeric glycoprotein found on the surface of many cells and in many extracellular fluids (including plasma), which can specifically bind fibronectin. Gelatin agarose gel 4FF is an affinity chromatographic packing specially designed for purification or removal of fibronectin.

1 Technical Specifications

2 Usage Method

2.1 Gel Preparation

(1) Let the gel and all the reagents reach the working temperature.

(2) Preparation of initial buffer (balance) and eluting buffer.

(3) Gelatin agar-gel 4FF is stored in 20% ethanol solution, take an appropriate amount of gel, clean 20% of the ethanol, and drain. Prepare a homogenate with a buffer (gel: buffer = 3:1 ratio).

2.2 Column Installation

(1) Wet the inside of the column and the bottom of the column with water or buffer and maintain a small level (the liquid level is slightly higher than the filter membrane), be sure to make the bottom no bubbles.

(2) Use a glass rod to guide the homogenate along the inner wall of the column into the column at one time, taking care not to produce bubbles. Open the liquid outlet of the column, make the gel settle freely in the column, and connect the top of the column.

(3) Open the peristaltic pump, so that the buffer flow rate of 1.33 times the flow rate of use, so that the column bed is stable. Balance the column with a buffer of 2 to 3 times the volume of the column.

2.3 Balancing Let the balancing buffer flow through the column at a certain flow rate until the effluent conductance and pH are unchanged.

2.4 Loading (1) The sample is prepared with a balanced solution, usually phosphate buffer or Tris-HCl buffer, and the turbid sample should be centrifuged and filtered before loading.

(2) In general, the target product is combined on the column, the impurities are washed with the balance solution, and then an eluent is selected to wash the target product.

2.4 Elution Gelatin-agar-gel 4FF can elute fibronectin in different ways:

(1) Use a buffer containing bromine salts, such as sodium bromide or potassium bromide, and elute at conditions below the pH of the equilibrium buffer. A recommended elution buffer is 0.05 M of sodium acetate, pH 5.0, and 1.0 M of sodium bromide or potassium bromide.

(2) 8 M of urea can be added to the equilibrium buffer to elute fibronectin adsorbated on the gel.

(3) Fibronectin can also be eluted by adding arginine to the equilibrium buffer.

2.6 Regeneration Depending on the nature of the sample, gelatin agarose gel 4FF can be regenerated to achieve the purpose of reuse. A buffer of high pH (0.1M Tris-HCl, 0.5M NaCl, pH 8.5) and a buffer of low pH (0.1M NaAC, 0.5M NaCl, pH 4.5) with a volume of 2-3 cylinders can be used alternately to clean the gel. One cycle is repeated 3 times, and then balanced with 3-5 times the column volume of the balance buffer, you can carry out the next test. If detergents or denaturants (such as 8 M urea) are used in the purification process, then these detergents can also be used in the elution buffer. 2.7 Cleaning In some applications, samples such as denatured proteins or lipids that are not eluted during the regeneration process can be washed for a while at 37℃by adding some detergent, such as 0.1% Triton X-100. Immediately re-balance with a combined buffer of at least 5 times the column volume. 2.8 Storage Gelatin-agar-gel 4FF should be stored in a 20% ethanol solution at 4-8℃. Special attention: the pigment must be removed from the sample before loading, otherwise the pigment will be adsorbed to the filler, affecting the normal use of the filler.

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