Gentle Elution Buffer

Elute the bound conjugate with 0.1 M glycine, 0.15M NaCl, pH 2.8, or another suitable elution buffer. A neutral pH alternative to this buffer is the Gentle Elution Buffer from Thermo Fisher. If acid pH conditions are used, immediately neutralize the fractions eluting from the column by the addition of 0.5 ml of 1M Tris, pH 8.0, per fraction. 

Gently homogenise the gel in a siliconized tube with a glass rod and cover the gel fragments with 0.5 ml of Elution Buffer (0.5 M-NaCl, 0.005M-EDTA, O.IM-Tris HC1, pH 8.5). Leave at 37°C for 5 hr or more. (Alternatively the procedure described in Section 5.9., procedure A, can be used.)... 

Gently resuspend the purification resin and if three columns will be run simultaneously, divide it equally into three beakers. Attach the provided fimnel to the column and carefully and evenly pour one-third of the resin into the column to form a densely packed resin bed. Allow the column to flow until the elution buffer stops at the top of the column (wNote 4). 

Elute protein by adding 50 pL of Ni-elution buffer. Mix gently. Incubate for 5-10 min at room temperature. Spin down at 420 x for 1 min. Remove 45 pL from the eluted protein and... 

Co-purifying proteins are eluted in 200 [A of buffer F containing 400 ng//(I FLAG peptide (Sigma) at room temperature with gentle rocking. 

If gentle and selective elution methods do not release the bound macromolecule, then mild denaturing agents can be added to the buffer. These... 

Thiol Sepharose 4B derivatized with moenomycin A was incubated with the preselected RNA pool for 1 h at room temperature while being mixed gently. This mixture was then transferred to an empty chromatography column. Unbound RNA was removed from the matrix by washing with selection buffer. Bound RNA was eluted by cleaving the moenomycin A from the Sepharose by washing the column 5 times with 200 pL selection buffer containing 200 mM DTT. 

---