Strep-TactinXT 4Flow columns contain a 4% agarose coupled with the streptavidin variant Strep-TactinXT and are applicable for purification of Strep-tagII or Twin-Strep-tag fusion proteins via gravity flow. Due to the broad pH compatibility, excellent pressure stability, outstanding target purity (> 95%), and highest binding affinity, the resin is recommended for all protein classes, especially for low abundant or challenging proteins. The low concentrated agarose allows for the simple attachment of larger proteins to the stationary phase, making it particularly suitable for purification of high-molecular-weight targets. In addition, Strep-TactinXT 4Flow enables the application of denaturing agents such as urea. To reduce the waiting period during the sample and buffer application, the use of the application aid WET FRED is recommended.