HisCube Ni-INDIGO His-Tag Protein purification MIDI protocol


Thank you for choosing our HisCube MIDI Kit, the superior alternative to traditional Ni-NTA based His-Tag protein purifications. This kit aims for the upmost purity and highest possible protein yield that IMAC based His-Tag purifications can achieve.

Components of the Kit

The components were of Kit were selected and developed under the main aspect of "efficiency". Meaning that an overloaded Kit with a magnitude of different buffers and working steps was avoided. Everything component (including the packaging) is designed to be as efficient and enviroment friendly as possible.

Table 1: Contents & general features of the Kit.

Feature 
Contents of the Kit 1 x 10 mL Ni-INDIGO agarose
2 x 50 mL Binding Buffer
2 x 50 mL Wash Buffer
1 x 50 mL Elution Buffer
500 mM EDTA (add 0.866 mL dH2O)
500 mM DTT (add 1.016 mL dH2O)
5 x MIDI Centrifuge Columns
Required Equipment Pipettes & matching tips
table centrifuge
1.5 ml tubes
Syringe + Filter for filtration
End-over-end shaker
optional: Protease Inhibitor
Storage Temperature 4°C / 39°F
Package This package is made up of 100% recycable material and produced according to FSC guidelines
Product ID 80101
Support https://cube-biotech.com/ Live Chat Support OR [email protected]

Information

The INDIGO-Ligand was developed by Cube Biotech and has proven numerous times to be the best option to reach maximum purity & protein yield simultaneously.

The PureCube Ni-INDIGO resin consist of 6% cross-linked agarose beads with an average diameter of 100 µm. The resin has a 50% concentration and a protein binding capacity of up to 100 mg / mL The provided MINI spin-columns are suited for laboratory centrifuges.

What sets this kit apart from other His-tag purification kits?

The novel feature that sets this Kit apart from traditional His-tag kits is our in house developed INDIGO-Ligand.

The key advantage of the INDIGO ligand compared to other options like NTA or IDA is that its capability to tolerate 20 mM EDTA and 20 mM DTT included in the purification buffers.

In comparison: NTA based His-tag purification products tolerate up to 10 mM DTT and 1 mM EDTA.
INDIGO Purification qualitiesFigure 1: Ni-INDIGO (left) resin compared to Ni-NTA from another competitor (right). This test compared the protein yields & purity of both resins using controlled amounts of his-tagged GFP in the corresponding cell lysates.

Contents of the Kit

  • 1-step batch Mini Columns: These columns retain resin and sample in chamber for batch incubation. By centrifugation, the membrane pores dilate and the filtered eluate gathers in the collection chamber of the column. The Mini batch spin columns are made for expression trials, small-scale screening and other small-volume purification needs.                                                                                                                                         
  • Buffers:The HisCube INDIGO-Ni Purification Kit contains three different buffers. Each buffer is optimized for our Ni-INDIGO beads. Note that the Elution Buffer contains 250 mM Imidazole. Please read the section “Protein quantification“ regarding the effect of imidazole on protein quatification and how to overcome it.                                                                                                                                                
  • EDTA & DTT : have great advantages in a lot of buffers involved in protein purification, but unfortunately ligands like NTA or IDA get stripped by them and therefore lose their functionality.                                                                                                                                                                                                             
    • DTT completed buffers can prevent protein aggregates since it dissolves disulfid bridges between proteins.
    • EDTA: can chelate cations that are co-factors of some proteases. Therefore it can function as a protease inhibitor. This results in higher proteins yields and protein stability. EDTA is mostly effective as part of the Binding buffer.
Note: DTT becomes instable after being dissolved in water. Only add it to the buffers directly prior to its use. Freeze the remaining stock at -20°C.

Both EDTA & DTT come lyophilized. Simply add the given volumes to the powders to get a 500 mM stock solution of each substance. Both their final concentration in the buffers should be around 1-5 mM.
  • Ni-INDIGO resin: The INDIGO ligand is a novel development of Cube Biotech and is the solution to a lot of problems that plague His-tag protein purifications.
    As figure 1 shows, His-tag purifications using the INDIGO ligand are much purer than traditional Ni-NTA.

    Ni-INDIGO resin beads are composed of 6% cross-linked DNA with an average bead diameter of 100 µm. It can be used in batch spin columns as demonstrated in this kit. Or in FPLC drip cartridges as part of our HisCube Ni-INDIGO His-tag Protein Purification MIDI Kit.

    It can bind up to 100 mg protein per ml resin used.
Ni-INDIGO agarose resin

Protocol

Every protein purification assay starts with the extraction of the protein of interest from the expression cell. This procedure is dependent on the used expression system. As already stated our Ni-INDIGO resin is compatible with protein lysates from E.coli, mammalian cell lines, insect cell and yeast to name a few. Possible mechanical disruption methods are freeze/thaw cycles, sonication, French press or glass bead lysis (yeast). Please refer to your own lysis protocol. For small scale purification we recommend sonication.
In before: Make sure to keep a sample of all your Elution, flow-through and Wash-fractions for analysis.
 
  • Step 1: Shake the Ni-INDIGO resin flask well to mix the settled beads with the storage buffer. If you are using EDTA or DTT add them now to the Binding, Wash and Elution Buffers.
  • Step 2: Place the MIDI centrifuge column in a conventional 50 mL (falcon) tube.
  • Step 3: Pipet 2 mL of the 50% Ni-INDIGO slurry into the batch spin column. Centrifuge the resin at 3000 x g for 20 seconds. Remove the flow-through.
Note: Step 3 is critical to ensure that all ethanol is removed from the resin to avoid interference with the membrane technology.INDIGO-Ni Storage buffer contains 20% Ethanol.
  • Step 4: Equilibrate the MIDI centrifuge column with 5 ml binding buffer by centrifuging the spin column at 3000 x g for 20 seconds. Remove the flow-through.
  • Step 5: Repeat step number 4 once to remove any residual ethanol.
  • Step 6: Immediately before loading re-filter the sample through a 0.2 µm filter (e.g. syringe filter) to remove any solid particle that might clog the column.
Note: It is critical to perform this step immediately before loading the sample on the column to ensure optimal performance.
  • Step 7: Load the required volume of filtered sample. The maximum sample volume is 10 mL. Close the lid and vortex for 15 seconds to mix the sample and the resin.
Optionally: Add protease inhibitor to your cell lysate to prevent protein degration.
  • Step 8: Alternative A: Repeat the vortexing every 15 min for 1 hour. In some circumstances, more than 1 hour batch incubation may be required. Repeat the vortexing every 30 min – 1 hour.
OR
  • Step 8: Alternative B: ncubate on an end-over-end shaker at room temperature for 30 min, or at 4°C for 1 h, depending on the temperature stability of the protein.
  • Step 9: Centrifuge the lysate at 3000 x g for 20 seconds. Save the flow-through. This flow-through is called flow-through fraction.
  • Step 10: Load the spin column barrel with up to 3 mL of Wash Buffer. Vortex or invert the closed tube for a couple of times. This spin at 3000 x g for 20 seconds. It is best to save the flow-through. This flow-through is called wash fraction.
Note: The flow-through contains the wash fractions. Keep aliquots of the individual wash fractions for subsequent SDS-PAGE analysis.
  • Step 11: Repeat the wash step for at least two times to ensure removal of unspecifically bound protein. If applicable, check the samples for protein content using a UV-spectrophotometer. Absorbance at 280 nm should be < 0.1.
  • Step 12: Elute the target protein by adding 0.6 - 6 mL Elution Buffer and centrifuging at 3000 x g for 20 seconds. If necessary, repeat the elution step up to 5 times.
Note: This 12-fold range of Elution Buffer volumes is meant to allow the adjustment of the final volume to the protein expression level. Low expressed proteins should not be eluted in a big volume.
  • Step 13: Save each eluate fraction inside the collection tube in a separate tube of appropiate size and determine the protein concentration by measuring absorbance at 260 - 280 nm.
Optional: Use a fresh tube for the elution step to avoid contamination with the previous wash fractions.

 

Quantification

It is important to remember that the elution buffer contains 250 mM Imidazole. This substance has its own absorbance at 280 nm, similary to proteins.

Therefore it must be removed in before. For this purpose we recommend to perform a dialysis of your eluated protein.

Now you can measure its absorbance at 280 nm.

Troubleshooting

In Advance: If your problem can not be solved by this troubleshooting guide, feel free to state your problem directly to: [email protected] or contact us via our chat support on our homepage!
  • Protein Yield: Problems with the protein yield can be numerous, follow this checklist to avoid the most commonly made ones.
    • Expression: Always confirm that your protein is indeed expressed. Check your lysate e.g. through a Coomassie stain, to confirm the presence of your protein of interest.
    • Expression II: Afterwards you should perform a westernblot with e.g. a Penta-His Antibody to exclude the possibility of a false-positive result of the Coomassie stain.
    • Tag Accessibility: Occasionally the 3D structure of a protein can mask the His tag and therefore hinder the Ni-INDIGO bead‘s access to the tag. There are two ways to prevent this: First change the terminus of the protein where the His-tag. is attached to. Second, add a spacer sequence between the protein‘s terminus and the His-tag.
  • Protein purity: Add additional washing steps, by additional repeats of step 10 or slightly increase the imidazole concentration of the wash buffer to get rid of unwanted molecules with less affinity to the beads. cell extract.
Note: Imidazole effects the pH of your buffer, check upon addition.
  • Column flow: Most of the time the column flow is hindered by cell debris that is left over in the cell lysate. Remember to follow step number 5 of the protocol to prevent this.
    A second reason can be the overload of the column. In this case add less cell extract.

  • Protein aggregates: Sometimes proteins can aggregate in a lysate. This prevents proper binding to the INDIGO beads. To counter this add around 1-5 mM DTT to all buffers.


  • Active proteases: Sometimes the protein of interest is degraded by proteases that can be found inside the cell lysate. These need to be inactivated. As some proteases usually work with cations as co-factors, EDTA will chelate these cations and thus inactivate the proteases.
    If this is the case we recommend to add 1-5 mM EDTA to the Binding buffer.
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