It may still work using your own amylose solution. However, there will probably be reduced performance (less binding) compared to the supplied amylose resin.

The following concentrations of commonly used chemicals have been tested with several different proteins and did not affect protein purification: Reducing Agents 50mM beta-mercaptoethanol Non-Ionic Detergents 2% Nonidet® P-40 2% Triton® X-100 2% Tween®-20 Ionic Detergents 0.1% SDS (Sodium-N-dodecyl sulfate) Denaturing Reagents 1 M Urea (Protein Dependent) Chemicals and other Reagents 50mM EDTA 10% (v/v) Ethanol 25% (v/v) Glycerol 250mM Imidazole 1M NaCl

pH 6-9 have been evaluated successfully with several different proteins, however this may vary depending on the specific protein. Lower and higher pH have not been tested yet.

No, this will prevent the protein from binding to the matrix.

Check your buffers for signs of contamination and check the pH of the buffers. - Increase centrifugation time and speed. Ensure that the Amylose Resin drains completely after each spin (some older centrifuge models may require a longer centrifugation time). - If the problem persists, additional wash steps can be added to the purification protocol.

Optimize the lysis buffer. Reducing the detergent concentration or using a different lysis buffer might enhance yield. - Optimize the growth medium used for protein expression. Adding glucose to the media at a final concentration of 0.2% represses the expression of amylase, which interferes with protein binding to the resin. - Optimize expression conditions. Try expressing the protein at lower temperatures to increase solubility. - Reload the column. Simply repeat steps 6 and 7 until the desired volume of sample has been loaded onto the column.

The starting material contains Maltose. Free Maltose binds to the Amylose Resin and thus reduces the binding capacity for the desired protein. Too much detergent is used. Nonionic detergents or other chemicals present in the crude extract can reduce binding affinity. Some fusion proteins bind less efficiently in the present of detergents than others. Growth medium used for protein expression contains too much amylase. Cells grown in LB and similar media have substantial amounts of an amylase, which interferes with binding. Expression of insoluble protein. Overexpression of proteins may result in the formation of insoluble inclusion bodies inside cells. If a large band of over-expressed protein is visible after SDS-PAGE electrophoresis of intact cells, but not present in the cleared cell lysates, then the expressed protein may not be soluble or form inclusion bodies. Starting material is too dilute. If the starting material contains very low levels of MBP-tagged protein, then it may require more than 800µl of sample volume to purify enough protein. Simply repeat steps 6 and 7 until the desired volume has been loaded onto the column.

In the Paper from Walker et. al. 2009 (file:///Y:/Zymo Research Europe/Labor/Projekte/Tag-Spin/MBP-Spin/Literature/Walker_2010.pdf), it was shown that the MBP containing certain mutations has an enhanced binding to maltodextrin. However, in our hands we could not confirm this result. When purifying MBP-fusion proteins with or without mutations with our kit there was no difference in yield.

We do not recommend using less than 50µl of MBP-Elution Buffer. Volumes less than this will not fully saturate the resin and result in reduced elution efficiency.