TITLE: Maranhao Polymerase/Protein Purification Protocol
AUTHORS: Andre Maranhao, Andrew  D Ellington

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">I present this as a somewhat universal/standard/basic purification protocol that has been successfully used to purify polymerases (e.g. RTX and Bst-LF) as well as other accessory proteins. Although this protocol was developed and codified for the purification of RTX (1), it has and can be used to purify a range of proteins of differing stabilities, solubilities, etc.</div></div>

[STEPS]
?. [Culture Centrifugation]
Spin down  culture  . Discard the supernatant (i.e. spent media). Keep cell pellet on ice or in a cold room enivronment  ().If working from a frozen pellet, simply thaw cell pellet on ice.
1 L
Centrifuge: 5000 34, 20 min, 4 10
4 °C
?. [Lysis]
Resuspend cell pellet in  Lysis Buffer and transfer to a clean 50 mL beaker with stir bar. Securely place the beaker setup into an ice bath.Sonicate with moderate spinning on ice at:    - 40% amplitude    - 1 sec ON / 4 sec OFF cycle    - 4 min total ON timeTransfer sonicated samples to clean, cold Nalgene Oak Ridge ultracentrifugation tubes.
30 ml
?. [Generating cleared lysate]
First Ultracentrifugation:Carefully balance all Oak Ridge tubes containing sonicated cell pellet.Centrifuge lysate .Following first ultracentrifugation, transfer supernatant to a new Nalgene Oak Ridge ultracentrifugation tubes and carefully rebalance all tubes.
Centrifuge: 35000 34, 30 min
?. [Generating cleared lysate]
Optional Heat-treament Step:NOTE: Skip this optional step if target protein is not thermostable or is not known to be thermostable.Otherwise, following first ultracentrifugation, place Nalgene Oak Ridge ultracentrifugation tubes into a thermomixer and shake at 400-800 rpm for :Set thermomixer to the appropriate temperature depending on target protein properties.KOD/RTX:  85°CBst-LF:  65°C After shaking at temperature, place Nalgene Oak Ridge tubes on ice for  prior to the second ultracentrifugation.
?. [Generating cleared lysate]
Second Ultracentrifugation:Centrifuge lysate at .Following this second ultracentrifugation, transfer supernatant to a 50 mL Falcon tubes and keep cold.
Centrifuge: 35000 34, 30 min, 4 10
?. [Generating cleared lysate]
Lysate Filtration:Filter resulting supernatant using a 0.2 μm filter. The resulting lysate is "Cleared Lysate" (CL).
?. [Ni-NTA Column Purification]
Apply Cleared Lysate (CL) to preassembled gravity flow columns containing properly equilibrated Ni-NTA. Collect all Flow-through (FT).Wash column with 20 column volumes (CVs) of Equilibration Buffer. Collect Wash 1 (W1).Wash column with 5 CVs of Wash Buffer. Collect Wash 2 (W2).Elute target protein with 5 CVs Elution Buffer.
?. [Ni-NTA Dialysis]
Perform all setup and the sequential dialysis in a cold room environment (4°C). Use a dialysis cassette of the appropriate volume and molecular weight cut-off (MWCO). Select a MWCO equal to or less than half the size of target protein.
?. [Ni-NTA Dialysis]
First, hydrate the dialysis cassette and its membrane in 2 L Ni-NTA Dialysis Buffer for at least 5 min. Next, fill the now hydrated dialysis cassette with eluate from Step 4, carefully remove air from the cassette, and allow to dialyze for 3-4 hours.
?. [Ni-NTA Dialysis]
Following initial dialysis, transfer cassettes from the first 2 L Ni-NTA Dialysis Buffer to a second volume of dialysis buffer and allow to dialyze overnight.
?. [FPLC Polishing]
Equilibrate a 5 mL Heparin column with Heparin Buffer A. Apply the overnight dialyzed eluate to equilibrated Heparin column. Elute along an NaCl gradient from 100 mM NaCl (Heparin Buffer A) to 2 M NaCl (Heparin Buffer B) collecting fractions along the salt gradient.Run a PAGE gel to verify the identity and relative purity of the peak presumed to be target protein.
?. [Final Dialysis]
Perform all setup and the sequential dialysis in a cold room environment (4°C). Use the same dialysis cassette selected for Setp 5.
?. [Final Dialysis]
First, hydrate the dialysis cassette and its membrane in 2 L Heparin Dialysis Buffer for at least 5 min. Next, fill the now hydrated dialysis cassette with the desired fractions from Step 6, carefully remove air from the cassette, and allow to dialyze for 3-4 hours.
?. [Final Dialysis]
Following initial dialysis, save an aliquot (~40 mL) of Final Heparin Dialysis Buffer to be used in dilutions of the final dialysis product. Transfer cassettes from the first 2 L Final Heparin Dialysis Buffer to a second volume of dialysis buffer and allow to dialyze overnight.
?. [Quantification]
Quantify protein concentration using the saved aliquot of Final Heparin Dialysis Buffer as a blank. Use that same aliquot of Final Heparin Dialysis Buffer to perform dilutions from the final/stock protein.