Dilution Preparative Membrane Protein Folding Kit (Condition #1)

Mfr.No.	MPP01
Description	ProFoldin Dilution Membrane Protein Folding Screen Kit (catalog # MPS01-20) provides 20 optimized conditions for screens of membrane protein folding conditions. About 70µg of urea-solubilized proteins from inclusion bodies are used for each condition. Once the folding conditions are identified, preparative folding kits (see catalog numbers above) are available for preparative scale folding. Each preparative folding kit is for folding 5 mg of urea[1]solubilized proteins. The condition number is identical to the Solution S number in the kit. The Dilution Membrane Protein Folding Screen Kit (catalog # MPS01-20) includes 0.4 ml of Reagent A, 0.4 ml of Reagent B, 7.2 ml of Reagent C, 25 ml of 20 Solution S.

Product Manual	Datasheet
Protocols Description	PROTEIN FOLDING PROCEDURE Folding Screen (1) Solubilize the inclusion bodies in 20 mM Tris-HCl, pH 7.0, 8 M urea, 10 mM DTT, 5 mM EDTA at room temperature for 4 hr. Centrifuge the solubilization material at 125,000 x g for 30 min to remove any insoluble materials. If the protein purity is < 50 %, purification of the denatured protein is recommended. Adjust the protein concentration to about 5 to 10 mg/ml. (2) For Conditions 1 to 10: Pre-incubate Solution S1 to S10 at 4ºC. Mix 105µl of the urea-solubilized protein with 55µl of Reagent A to make Sample A. Incubate Sample A at room temperature for 2 hr. Then mix 15µl of Sample A with each Solution S1 to S10 (350µl) at 4ºC for 2 hr. For Conditions 11 to 20: Warm Reagent B to room temperature to solubilize any precipitates. Pre-incubate Solution S11 to S20 at room temperature. Mix 105µl of the urea-solubilized protein with 55µl of Reagent B to make Sample B. Incubate Sample B at room temperature for 2 hr. Then mix 15µl of Sample B with each Solution S11 to S20 (350µl) at room temperature for 2 hr. (3) Add 100µl of Reagent C into each folding solution and incubate all solutions at 4ºC overnight. (4) Spin the solutions at 14,000 rpm for 10 min and collect the supernatant for analysis of the folded protein. Preparative folding Use the optimized condition to scale-up the folding reaction. Follow the same procedure as that in the screen experiment but in a larger volume (70 fold) of solutions: 700µl of urea-solubilized protein; 350µl of Regent A (for the following protocols of protein inclusion body preparation and solubilization are recommended). Inclusion body isolation: 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. Increase the volume proportionally for cell pellets from more than 1 L of culture. 2. Break the cells by passing the cell suspension French Press twice. 3. Centrifuge the broken cell suspension at 20,000 rpm for 20 min. 4. The crude inclusion bodies are in the pellet. Inclusion body purification: 1. Resuspend the crude inclusion bodies in the cell lysis buffer plus 1 % Triton-100 by stirring at 4°C for 1 to 2 hours. 2. Centrifuge the suspension at 20,000 rpm for 20 min. Discard the supernatant. 3. Resuspend again the pellet in the cell lysis buffer without Triton. 4. Centrifuge the suspension at 20,000 rpm for 20 min. Discard the supernatant. The pellet is the purified inclusion bodies. Inclusion body solubilization: 1. Estimate the amount of protein in the purified inclusion bodies. Add the solubilization buffer (20 mM Tris-HCl, pH 8.0, 8 M urea, 10 mM DTT) to make about 10 mg/ml protein concentration. The solubilization is performed by stirring the inclusion bodies with the solubilization buffer at room temperature for 2 to 4 hours. Most of the pellet should be solubilized. 2. Centrifuge the solubilization material at 30,000 rpm for 45 min. Save the supernatant as the solubilized inclusion bodies.

Have you used this product?

Rate it now..!!

Combination SKU	Supplier No.	Specifications	Your price
BHE16300305-1	MPP01	Condition #1	$303.18	Add to Cart
BHE16300305-2	MPP10	Condition #10	$303.18	Add to Cart
BHE16300305-3	MPP11	Condition #11	$303.18	Add to Cart
BHE16300305-4	MPP12	Condition #12	$303.18	Add to Cart
BHE16300305-5	MPP13	Condition #13	$303.18	Add to Cart
BHE16300305-6	MPP14	Condition #14	$303.18	Add to Cart
BHE16300305-7	MPP15	Condition #15	$303.18	Add to Cart
BHE16300305-8	MPP16	Condition #16	$303.18	Add to Cart
BHE16300305-9	MPP17	Condition #17	$303.18	Add to Cart
BHE16300305-10	MPP18	Condition #18	$303.18	Add to Cart
BHE16300305-11	MPP19	Condition #19	$303.18	Add to Cart
BHE16300305-12	MPP02	Condition #2	$303.18	Add to Cart
BHE16300305-13	MPP20	Condition #20	$303.18	Add to Cart
BHE16300305-14	MPP03	Condition #3	$303.18	Add to Cart
BHE16300305-15	MPP04	Condition #4	$303.18	Add to Cart
BHE16300305-16	MPP05	Condition #5	$303.18	Add to Cart
BHE16300305-17	MPP06	Condition #6	$303.18	Add to Cart
BHE16300305-18	MPP07	Condition #7	$303.18	Add to Cart
BHE16300305-19	MPP08	Condition #8	$303.18	Add to Cart
BHE16300305-20	MPP09	Condition #9	$303.18	Add to Cart

Dilution Preparative Membrane Protein Folding Kit (Condition #1)

Contact us to order

Tel

Email

Contact us to order

Tel

Email

Reviews of Dilution Preparative Membrane Protein Folding Kit (Condition #1)

Rate it now..!!