(1:1 suspension in a 20% ethanol solution)
pkg of 1 mL
pkg of 100 mL
pkg of 25 mL
pkg of 5 mL
pkg of 500 mL
1.5-2.4 mL/mL (suspension in packed gel)
protein purification: suitable
faint blue to very dark blue
6% Beaded Agarose
>15 mg/mL, gel binding capacity (protein)(with an approx. 30 kDa protein)
flash point 32 °C (closed cup)
UN1170 - class 3 - PG 3 - Ethanol, solution
89.6 °F - closed cup
32 °C - closed cup
For column chromatography, no more than 20 mM is suggested in the extract, equilibration, and wash buffers to prevent non-specific binding of proteins. No more than 250 mM is suggested for the elution buffers. Many proteins will elute with imidazole levels as low as 100 to 200 mM. For batch methods the imidazole concentration may have to be reduced or eliminated.When a protein is expressed at low levels, the presence of the imidazole limits the binding of the protein in the batch method but not when used in a column.
Yes, Tris buffers should work.
Verify the pH and composition of sample and equilibration buffers. Make sure there are no chelating or reducing agents present in the extraction buffer. If using the batch mode, remove imidazole. Run the affinity purification under denaturing conditions. Run a Western blot of the extract to verify that the recombinant protein is present.
0.1% SDS has been used with some samples, with no adverse effects on the observed protein binding. However, SDS will effectively coat proteins and may block the binding to the column. It is probably very protein-specific and an SDS concentration that works for one protein may not work for another.
During purification many protein extracts tend to discolor an affinity gel during the loading step. The original color will return after the wash or elution step. If the color is still not changing strip and recharge the affinity gel with nickel. Wash with EDTA and recharge with Nickel solution.
If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis.
The lot specific COA document can be found by entering the lot number above under the "Documents" section.
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