Gel layering method

1 . Gel SelectionChoose different models of gels based on the molecular weight of the layered substance, such as desalination and free fluorin, with the option of coarse, medium-grained G
28 or G
500 ,G
250used to isolate
protein
monosomes, G
200more used to isolate protein gel polymers and so on.2 . Pre-treatment of the gelsaid that the appropriate amount of gel added to the excess buffer in the refrigerator (or room temperature) fully expanded, or boiled in boiling water, the expansion time should be based on different types of gel. In order to make the particles uniform and consistent need to be flotation, that is, after adding gel particles, gently stir, sit 20min , dump the precipitated particles, so repeated several times can be.3 . The choice of
column is generally based on the type of sample isolated and the number of samples. When purifying protein, the column bed volume should be25100sample volume. The salt-free luciferin is about 4 to 10 timesthevolumethe samplevolume. The bar is too short to affect the separation effect. The column is longer, the separation effect is good, but the column is too long, the separation time is prolonged, and the sample is diluted too much. The inner diameter of the layering column should also be selected appropriately. The inner diameter is too fine, and a "wall effect" occurs, i.e. the flow rate near the pipe wall is greater than the flow rate of the center affects the separation effect. Therefore, the inner diameter and height of the column should have a certain proportion. For desalination should be 1 . 5to 1 . 25 ; for purified proteins should be 1 . 20 to 1 . 100 . The column loading process is basically the same as the ion exchange analysis column. 4 . Samples and wash-out
sample volume should not be too much, preferably for the bed volume of 1% to 5% , not more than 10% . Sample concentration should not be too large, concentration is too large viscosity, poor separation effect, generally not more than 4% . The sealing should be consistent with expansion, otherwise the solvent will be replaced and the volume of the gel will change, affecting the separation effect. The sealing should have a certain ion strength and pH value. Separation

0.02 to 0.1Mol/L pH 6.9 to 8.0 PBS (0.14Mol/L NaCl ) and 0.1Mol/L pH8.0Tris-HCl buffer salt solution (0.14Mol/L Nacl ). 5 . The sealized liquid
and ion exchange layer analysis. 6 . Re-use and preservation of the gel column when all the parts of the sample have been washed off, a new sample can be added and continued to be used. There are three ways to save: (1) is most convenient to keep in the liquid phase, i.e.
preservatives
(usually 0.02% N) in the gel suspension
Save
2 N
3 or 0.002% Washing Pizza) or
Autoculsion
4 degrees C. This method can be saved for at least half a year. (2) after has run out, rinse with water, then rinse with 60% to 70% alcohol, the gel volume is reduced, i.e. stored in a semi-shrinking state. (3) long-term use, it is best to
dry
state, that is, after washing, with ethanol-containing water washing, gradually increase the amount of ethanol, and finally with 95% of ethanol washing, can be completely dehydrated, and then use ethylene to remove ethanol, filter dry, in 60 degrees C to 80 degrees C after drying.