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ProteoLiposome BD Expression Kit

Product Name Catalog No. Qty No. of Reaction Storage
ProteoLiposome BD Expression Kit CFS-TRI-PLE-BD 1 6 (*) -80

*Kit provides additional reagents for testing expression vectors in the absence of liposomes.

※This kit does not include dialysis device. Recommend to use Thermo Scientific™, Slide-A-Lyzer™ MINI Dialysis Devices, 10K MWCO

Details

CellFree Sciences developed a new reaction BD (“bilayer and dialysis”) reaction format for higher-yield preparation of proteoliposomes, which combines our bilayer protein expression method with a dialysis reaction. During our tests on the expression of a number of membranes proteins, we found that the new BD reaction format yielded about up to four times the protein amounts of a standard bilayer reaction. This new method is suitable to obtain for many membrane proteins the necessary protein yields for antigen preparation. The reaction conditions used in this kit have been tested for the expression of various membrane proteins, where a 2.5 ml BD expression reaction yields for example for the G Protein-Coupled Taste Receptor T1R1 over 500 µg of protein in the purified proteoliposome fraction.

The ProteoLiposome BD Expression Kit provides all necessary reagents to perform six preparative 2.5 ml BD reactions. For easy use, the kit contains lyophilized liposomes prepared from asolectin from soybeans. After rehydration those liposomes can be directly used in the translation reaction, thus avoiding organic solvents involved in classical liposome preparation steps.

 

Figure 1: Illustration of proteoliposome preparation

  1. Dialysis device comprising 50 ml tube, dialysis cup, and lid (Thermo Scientific™, Slide-A-Lyzer™ MINI Dialysis Devices, 10K MWCO)
  2. Prepare tube and add feeding buffer
  3. Insert dialysis cup into tube with feeding buffer
  4. Add feeding buffer into dialysis cup within the tube
  5. Setup bilayer reaction in the dialysis cup within the tube. Refer to drawings within the orange box for more details on how to place the translation mixture below the feeding buffer
  6. Put lid onto tube with dialysis cup and conduct translation reaction
  7. Proteoliposomes will be formed within the dialysis cup

Figure 2: Comparison data between 4 ml Bilayer and 2.5 ml Bilayer-Dialysis translation

BL: Bilayer
BD: Bilayer-Dialysis
Sample :1 ul/lane
*: Detected protein

Lane No.

Sample Name

Protein Yield (ug)

Bilayer

Bilayer-
Dialysis

1 CHRM2 78 163
2 GHSR 156 539
3 PTGER1 121 293
4 GHRHR 165 767
5 GPR56 236 669
6 FZD7 235 514
7 GABBR1 167 552
8 T1R1 161 520
Kit Contents
Reagent name Qty Concentration Volume Box color Storage
WEPRO®7240 1 240 OD 1 ml White -80
5x Transcription Buffer LM 1 5x 0.4 ml
NTP Mix 1 25 mM 0.2 ml
SP6 RNA Polymerase 1 80 U/μl 30 μl
RNase Inhibitor 1 80 U/μl 30 μl
Creatine Kinase 1 20 mg/ml 20 μl
pEU-E01-T1R1 plasmid 1 1 mg/ml 30 μl
SUB-AMIX® SGC S1 1 40x 12.5 ml White -20
SUB-AMIX® SGC S2 1 40x 12.5 ml
SUB-AMIX® SGC S3 1 40x 12.5 ml
SUB-AMIX® SGC S4 1 40x 12.5 ml
Asolectin Liposome, lyophilized 6 (10 mg) Lyophilized Green -80
Instruction Manual
Reference
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