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Large-Scale Preparative Protein Folding Column Set

BHE16300367

This product includes 4 columns #1, 5.4 ml of Solution A, 15.4 ml of Solution C and 8.4 ml Solution B.

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Product Manual Datasheet
Protocols Description PROTEIN FOLDING PROCEDURE
Inclusion body preparation and solubilization
1. Resuspend the cell pellet in 20 ml of cell lysis buffer (20 mM Tris-HCl, pH 8, 100 mM NaCl, 2 mM DTT, 2 mM EDTA) for each liter of culture.
2. Break the cells by passing the cell suspension French Press twice and centrifuge the broken cell suspension at 20,000 rpm for 20 min.
3. Resuspend the pellet in the cell lysis buffer plus 1 % Triton-100 by stirring at 4°C for 1 to 2 hours and centrifuge the suspension at 20,000 rpm for 20 min. Discard the supernatant.
4. Wash the pellet in the cell lysis buffer without Triton by suspension and centrifugation.
5. Solubilize the inclusion bodies by stirring the pellet in 20 mM Tris-HCl, pH 8.0, 6 M guanidine hydrochloride (or 8 M urea), 10 mM DTT at room temperature for 2 hours. Then centrifuge the solubilization material at 30,000 rpm for 45 min. Save the supernatant as the solubilized inclusion bodies.
 
Protein folding using the large-scale preparative protein folding columns
The columns and reagents are cooled to 2 - 8°C. The experiment is performed in a cold room. An easy way to set up the columns is to use a test tube rack to hold the columns and place a tip box cover under the rack to receive the solution from the columns.
1. Sample preparation
To make the loading sample, mix 5 ml of the solubilized inclusion bodies with 5 ml of Solution A. Incubate the mixture (the loading sample) for 5 min.
2. Column preparation
Cut off the column bottom pointing tips and let the buffer run through the columns.
3. Protein folding
(1) load 2.5 ml of the loading sample per column. Let the sample completely run into the columns.
(2) Elute the protein with 3.5 ml of Solution C per column. The solution C number matches the column number. Collect and incubate the eluent at 4ºC for 2 to 4 hr. Discard the columns.
(3) Mix 8 ml of Solution B(a) with the 14 ml of eluent from the 4 columns and incubate the solution at 4ºC for 2 hr to overnight. Remove the precipitate (if there is any) by centrifugation. Note: If solution B forms precipitate during storage, warm it to room temperature to solubilize the precipitate, then cool it back to 4ºC before use.
 
PROTEIN PURIFICATION AFTER FOLDING
The folded protein can be purified by affinity, ion-exchange or gel filtration column chromatography. Some proteins are sensitive to low salt. To be cautious, brief dialysis of the protein solution against a buffer with a moderate salt concentration at 4ºC is recommended. Following is a Q-Sepharose column purification protocol:
(1) Dialyze the protein solution against 40 volumes of 20 mM Tris-HCl, pH 8.5, 50 mM NaCl, 2 mM DTT, 2 mM EDTA buffer at 4ºC for 2 to 4 hr. Remove any precipitates by centrifugation.
(2) Equilibrate a Q-Sepharose column with a low salt buffer (the same as the dialysis buffer). Load the dialyzed protein solution. Wash the loaded column with 10 column volumes of the same buffer. Elute the protein with a salt gradient.
(3) Any further protein purification step may follow as purification of regular soluble native proteins.
Combination SKU Supplier No. Specifications Your price Quantity
BHE16300367-1 PFC01 column #1 $352.55
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BHE16300367-2 PFC10 column #10 $352.55
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BHE16300367-3 PFC02 column #2 $352.55
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BHE16300367-4 PFC03 column #3 $352.55
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BHE16300367-5 PFC04 column #4 $352.55
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BHE16300367-6 PFC05 column #5 $352.55
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BHE16300367-7 PFC06 column #6 $352.55
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BHE16300367-8 PFC07 column #7 $352.55
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BHE16300367-9 PFC08 column #8 $352.55
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BHE16300367-10 PFC09 column #9 $352.55
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