His MultiTrap™ HP and His MultiTrap™ FF are prepacked, disposable 96-well ﬁlter plates for reproducible, high-throughput screening of histidine-tagged recombinant protein expression. Typical applications are expression screening of different constructs, screening for solubility of proteins, and optimization of the conditions for small-scale parallel puriﬁcation. The plates are prepacked with precharged Ni Sepharose® High Performance and Ni Sepharose® 6 Fast Flow, respectively.
Figure 3.6His MultiTrap™ HP and His MultiTrap™ FF
Each well of the prepacked His MultiTrap™ HP and His MultiTrap™ FF contains 500 µl of a 10% slurry of Ni Sepharose® High Performance or Ni Sepharose® 6 Fast Flow in storage solution (50 µl of medium in 20% ethanol) and has a capacity for purifying up to 1.0 mg and 0.8 mg of histidine-tagged protein, respectively. The plates are made of polypropylene and polyethylene.
Characteristics of the chromatography media and of His MultiTrap™ HP and His MultiTrap™ FF are listed in Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose® excel, TALON® Superflow, and uncharged IMAC Sepharose® products). The Ni2+-charged media are compatible with all commonly used aqueous buffers, reducing agents, denaturants, such as 6 M Gua-HCl and 8 M urea, and a range of other additives. Prepacked His MultiTrap™ HP and His MultiTrap™ FF plates provide well-to-well and plate-to-plate reproducibility in terms of yield and purity of eluted protein. Automated robotic systems can be used, as well as manual handling using centrifugation or vacuum pressure. The puriﬁcation procedure can easily be scaled up because Ni Sepharose® is available in both larger prepacked formats and as lab packs. This allows screening using His MultiTrap™ plates followed by scale-up on a HisTrap™ 1 ml or 5 ml column using best conditions, which shortens optimization time.
His MultiTrap™ HP: refer to Purification using Ni Sepharose® High Performance earlier in this chapter for a general procedure for sample and buffer preparation.
His MultiTrap™ FF: refer to Purification using Ni Sepharose® 6 Fast Flow earlier in this chapter for a general procedure for sample and buffer preparation.
After thorough cell disruption, it is possible to apply unclariﬁed lysate directly to the wells without precentrifugation and/or ﬁltration of the sample.
Apply the unclariﬁed lysate to the wells directly after preparation, as the lysate may precipitate unless used immediately or frozen and thawed before use. Samples with precipitation may be sonicated to reduce clogging of the wells. Note that aging of the sample may reduce yields of the target protein.
Lysis with commercial kits could give large cell debris particles that may interfere with drainage of the wells during puriﬁcation. This problem can be solved by centrifugation or ﬁltration of the sample before adding it to the wells.
Preparing the 96-well ﬁlter plate
Blank run: Reducing agents may be used in sample and buffers. In such a case, perform a blank run by applying 500 µl of elution buffer/well before step 7. No reducing agent should be used in buffer during blank runs. Reequilibrate with binding buffer that includes reducing agent before sample application. Do not leave His MultiTrap™ plates with buffers that include reducing agents when not in use.
Do not apply more than 700 × g during centrifugation.
If problems with foaming, reproducibility, or bubbles in the collection plate occur using vacuum, the centrifugation procedure should be considered. The distance between the ﬁlter plate and the collection plate is critical; reduce the distance to 5 mm if necessary.
Preparing the 96-well ﬁlter plate
Blank run: Reducing agents may be used in sample and buffers. In such a case, run a blank run by applying 500 µl of elution buffer/well before step 7. No reducing agent should be used in buffer during blank runs. Reequilibrate with binding buffer that includes reducing agent before sample application. Do not leave His MultiTrap™ plates with buffers that include reducing agents when not in use.
Do not apply a pressure in excess of -0.5 bar during vacuum operation.
If a robotic system is used for puriﬁcation, the vacuum must be adjusted according to methods applicable to the system.
Increasing the vacuum too quickly can result in foaming under the ﬁlter plate and subsequent cross-contamination of samples.
Determining solubility effects of detergents in buffers during puriﬁcation of membrane proteins using His MultiTrap™ FF
The 96-well ﬁlter plate format of His MultiTrap™ FF and His MultiTrap™ HP allows high-throughput screening and puriﬁcation of histidine-tagged proteins. In this example, His MultiTrap™ FF was used to screen eight detergents for their effect on the solubility of six histidine-tagged membrane proteins. Results from puriﬁcation screening of two proteins, GlpG protein (EM29) and cation transporter (EM43), are shown in a dot blot and SDS-PAGE in Figure 3.7. The results show that conditions to ﬁnd the most appropriate detergent for the membrane proteins in the study can be readily optimized, with high reproducibility, using MultiTrap™ 96-well ﬁlter plate.
Figure 3.7(A) Dot blot of membrane proteins EM29 and EM43 puriﬁed on His MultiTrap™ FF in the presence of different detergents. Repeats of eluates 1 and 2 shown in the dot blot are two independent extractions and puriﬁcations. (B) SDS-PAGE (Coomassie staining) of EM29 puriﬁcations (elutions 1 and 2 in the blot) with eight different detergents on His MultiTrap™ FF. Data kindly provided by S. Eshagi, V. Lieu, P. Nordlund, Dept. of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.