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Home / Assay Kits / Protein Science Assay Kits

Spin-column Protein Folding Screen Kit

BHE16300366

This product includes 10 spin columns #1-10, 160 ul of Solution A and 600 ul of Solution B.

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Reference 1. V. Ekaterina Kuznetsova et al. Genome-wide Analysis of Substrate Specificities of the Escherichia coli Haloacid Dehalogenase-like Phosphatase Family, J. Biol. Chem. VOL. 281, NO. 47, pp. 36149–36161 (2006).

2. Jooyoung Cha et al. Characterization of the β-Lactam Antibiotic Sensor Domain of the MecR1 Signal Sensor/Transducer Protein from Methicillin-Resistant Staphylococcus aureus, Biochemistry, 46 (26), pp 7822–7831 (2007).

3. XIE Hao et al. Overexpression and Purif ication of Membrane Transport Protein with Native Functions, Chinese J. Bioch. Mol. Biol. 23:12, 1051-1058 (2007).

4. Jacob Thomas et al. Acyl Carrier Protein Phosphodiesterase (AcpH) of Escherichia coli Is a Non-Canonical Member of the HD Phosphatase/Phosphodiesterase Family Biochemistry, 46, 129-136 (2007).

5. Jooyoung Cha et al. Lysine N-Decarboxylation in the BlaR1 Protein from Staphylococcus Aureus at the Root of Its Function As an Antibiotic Sensor J. Am. Chem. Soc., 129 (13), pp 3834–3835 (2007).
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 screen using the spin column kit
The columns and reagents are cooled to 2 - 8°C. The experiment is performed in a cold room.
1. Mix 130 µl of the solubilized inclusion bodies with 130 µl of Solution A. Incubate the mixture (the loading sample) for 5 min.
2. Spin the columns at 3200 rpm for 1 min using a bench-top microcentrifuge to set down the resin. Remove the column bottom tips and caps. Place the columns into 1.5 ml-microcentrifuge tubes and spin the columns at 1400 rpm for 2 min. Transfer each column into a clean labeled 1.5-ml microcentrifuge tube.
3. Load 25 µl of the loading sample onto each column and spin the columns at 3200 rpm for 4 min. Discard the columns and incubate the eluent at 4ºC for 2 to 4 hr.
4. Mix 50 µl of Solution B with the eluent from each column and incubate the solution at 4ºC for 2 hr to overnight. Spin the solution at 14,000 rpm for 5 min and collect the supernatant for analysis.
 
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.
 
ANALYSIS OF THE FOLDING PRODUCT
SDS-PAGE
Use SDS-PAGE to check protein solubility under each folding condition. To make the SDS-PAGE samples, mix 10 µl of the folding product from each column with 10 µl of water and 7 µl of 4 x SDS-PAGE loading buffer.
 
Activity test
Use an activity assay (catalytic or binding activity) to check the protein activity. Make 10 to 20 fold dilution of the folding product in the assay. For example, if the assay reaction volume is 100 µl, add 5 µl to 10 µl of the folding product for each reaction.
Combination SKU Supplier No. Specifications Your price Quantity
BHE16300366-1 SFC01-10 screen kit $183.66
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BHE16300366-2 SFC01 column #1 $147.02
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BHE16300366-3 SFC10 column #10 $147.02
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BHE16300366-4 SFC02 column #2 $147.02
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BHE16300366-5 SFC03 column #3 $147.02
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BHE16300366-6 SFC04 column #4 $147.02
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BHE16300366-7 SFC05 column #5 $147.02
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BHE16300366-8 SFC06 column #6 $147.02
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BHE16300366-9 SFC07 column #7 $147.02
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BHE16300366-10 SFC08 column #8 $147.02
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BHE16300366-11 SFC09 column #9 $147.02
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