Biogel protein separations

Gel filtration media such as Biogel P-30 (fine or medium) or Biogel P-6 (fine or medium Bio-Rad Laboratories Hercules, CA) Sephadex G-10, G-15, or G-25 (Amersham Biosciences Piscataway, NJ) or equivalent matrices made by many other manufacturers are typically used. For optimum separation of the biotinylated antibody from unattached label, researchers should choose a gel matrix with an exclusion limit smaller than the molecular weight of the protein sample. Thus, the protein will elute in the void volume of the column while smaller unwanted components will be strongly retarded by the matrix. Neither biotin nor antibody molecules are colored, so the progress of the gel filtration column must be monitored by absorbance at 280 nm. Small quantities of biotinylated antibodies can be... 

Resin (Bio-Rad BioGel P-30 Pine Size purification resin) is designed to separate free dye from proteins with MW > 40,000. Make sure the buffer elutes through the column with a consistently even flow prior to adding the reaction mixture. If the flow of the buffer is slow or stalled, repack the column. 

The CCC fractions, HDL-LDL and VLDL—serum proteins, were each separately dialyzed against distilled water until the concentration of potassium phosphate was decreased to the level in the starting buffer used for the hydroxyapatite chromatography. These two fractions were concentrated separately by ultrafiltration. The concentrates of both fractions were chromatographed on the hydroxyapatite column. Fig. 4 shows the elution profile on hydroxyapatite obtained from the HDL-LDL fraction. A 1.4 ml volume of the concentrate was loaded onto a Biogel HTP DNA-grade column (5.0 X 2.5 cm I.D.) and eluted at 1.0 mPmin with 75 and 290 roM potassium phosphate buffer at pH 7.4. Two lipoprotein peaks were eluted the first peak contained HDLs and the second peak contained LDLs. 

---