Use of organic solvents for protein purification from HIV-1 infected human plasma

doi:10.21203/rs.2.12144/v1

Method Article

Use of organic solvents for protein purification from HIV-1 infected human plasma

https://doi.org/10.21203/rs.2.12144/v1

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Human plasma contains various high molecular and low molecular proteins like Albumin, Globulin, Lipoproteins, Glycoproteins etc. To get a pure protein of interest , the organic solvents are very useful in the plasma protein purification steps. To achieve a low cost and reliable purification, the organic solvents are best useful in laboratory practice. Tri-choloroacetic acid and Acetone removes the Albumin and Globulin while Chloroform, methanol, isopropanol removes the Glycoproteins and Lipoproteins and would give the protein of interest in HIV-1 infected human plasma.

Biochemistry

TCA

Acetone

Chloroform

Methanol

Isopropanol

Plasma

SDSPAGE

HIV-1 infected human plasma contains Albumins, Globulins, Tri-glycerides, Phospholipids, Lipo-proteins etc. The HIV-1 infected patients those who were taking first line anti-retroviral therapy must have drug concentration in plasma. The removal of high abundant proteins like Albumin and Globulin, drug soluble proteins ,spingolipids, glycolipids, phospholipids are very essential from HIV-1 infected human plasma. Several methods like chromatographic methods, several manufacturer high abundant depletion kit etc. has been used to purify the proteins from human plasma. Butanol and Di-isopropyl ether was used for delipidation of plasma or serum without protein denaturation.¹Separation of plasma lipids also achieved by thin layer chromatography.²High density lipoprotein was fractionated from human plasma by gel filtration chromatography^3.The lipid also removed from human plasma by using cleanascite HC, a commercial product.⁴The lipoproteins were isolated from human plasma using the unltracentrifugation in zonal rotors.⁵Human plasma contains rich in glycoproteins. The glycoproteins from plasma was purified using the multilectin affinity column chromatography⁶. This protocol is low cost based on various organic solvents for purifications of several glycoproteins, lipoproteins, Albumins and globulins. The reason is to use different organic solvents as each organic solvent has the main role to remove lipoproteins, glycoproteins, albumins, globulins etc. Use of the mixture of organic solvents in a ratio removed the abundant proteins that might be interfering in 2-D gel electrophoresis, ELISA etc.

1. Chloroform( Merck,India)

2. Methanol(Merck, India)

3. Isopropanol (Merck, India).

4. TCA (Trichloro acetic acid, Sigma-Aldrich, USA)

5. Acetone( Mecrk, India)

6. Urea (Sigma-Aldrich, USA)

7. Thio Urea( Sigma-Aldrich, USA)

8. CHAPS(Sigma –Aldrich, USA)

9. ASB-14( Merck ,India)

10. Dithithretol( Sigma-Aldrich, USA)

11. Bromophenol blue (Merck, India, Cat.no:67-64-1).

12. H2O (HPLC purified ,Merck, India)

13. Acrylamide( Sigma –Aldrich, USA)

14. Bis-acrylamide(Sigma-Aldrich, USA)

15. Tris base( SRL,India)

16. Hydrochloric acid(Merck, India, Cat. No:1.09063.1000)

17. Sodium dodecyl sulfate(Himedia, India)

18. Ammonium per sulfate( Sigma-Aldrich, USA)

19. TEMED( Sigma-Aldrich, USA)

20. Glycerol( Merck,India, Cat No.56-81-5)

21. β-Mercaptoethanol( Sigma-Aldrich, USA)

22. Coomassiae brilliant blue-R250( Merck, India)

23. Acetic acid(Merck, India, Cat No. 64-19-7)

24. Methanol(Merck,India, Cat No.67-56-1)

1. -20 ^0CRefrigirator (Krispcold, India )

2. Centrifuge tube(Sigma G 3-18K)

TCA/Acetone and Chloroform: Methanol: Isopropanol(2:1:1) method of plasma protein purification:

1.200 µl plasma was taken in an eppendrof tube and 1mL of Chloroform: Methanol: Isopropanol( 2:1:1) was added and kept it for 15 minutes.

2. The tube was centrifuged at 12,000 rcf for 10 minutes at 4℃ and the supernatant was discarded.

3. Then, 800µl TCA/Acetone (50%) was added and incubated overnight at-20℃.

4. The tube was centrifuged at 12,000 rcf for 10 minutes at 4℃ and the supernatant was discarded.

5. 40% Ammonium sulphate was added to it and kept it for half an hour in ice .

6. Then, the tube was centrifuged at 12,000 RCF for 10 minutes and the supernatant was discarded.

9. Then the sample was desalted and concentrated using the 3Kd cut off column.

10. Then the pellet was cleaned up by Bio-rad clean up kit.

11. The pellet was dissolved in 150-250µl Rehydration buffer.

12. The sample was resolved through 10 % SDSPAGE.

The removal of chloroform, methanol, isopropanol phase is very important before adding acetone, a little disturbance to the tube created the turbidity of whole sample.

The time taken for the procedure would be 17 hours.

SDS PAGE analysis indicates that the use of various organic solvents like chloroform, methanol, iso-propanol in a ratio removes the lipoproteins and glycoproteins from HIV-1 infected human plasma initially. Then after TCA/Acetone overnight treatment removes the albumins and globulins from human plasma. So the plasma protein purification protocol is useful in 2-D gel electrophoresis , ELISA or any diagnostic based approach. This protocol is suitable for protein extraction from human plasma.

1.Cham, B. E. & Knowles, B. R. A solvent system for delipidation of plasma or serum without protein precipitation.J. Lipid Res.17, 176–81 (1976).

2. Brewster M &Quittner, H. Separation of Plasma Lipids by Thin-Layer Chromatography.Ann clin lab sci. 3, 79-84(1973).

3. Gordon, S. M., Deng, J., Lu, L. J. & Davidson, W. S. Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography. J. Proteome Res.9, 5239–5249 (2010).

4.Castro, A. R., Morrill, W. E. & Pope, V. Lipid Removal from Human Serum Samples. Clin.Vaccine Immunol.7, 197–199 (2000).

5.Wilcox, H. G., Davis, D. C. &Heimberg, M. The isolation of lipoproteins from human plasma by ultracentrifugation in zonal rotors.J. Lipid Res.12, 160–72 (1971).

6. Yang, Z., Hancock ,W.S., Chew, T.R. , Bonilla, L. A study of glycoproteins in human serum and plasma reference standards ( HUPO ) using multilectin affinity chromatography coupled with RPLC-MS / MS . Proteomics, 3353-66, 5(13):(2005).

Indian Council of Medical Research is acknowledged for providing the ICMR-SRFship to Mr Sushnata Kumar Barik.

All authors have no conflict of interest. Ethics approval: This study protocol was approved by the institutional ethics committee under guideline of ICMR, Govt. of India. All biosafty measures for handling HIV samples were followed as per the guidelines laid by Department of AIDS Control, India

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Use of organic solvents for protein purification from HIV-1 infected human plasma

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Use of organic solvents for protein purification from HIV-1 infected human plasma

Status:

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Abstract

Figures

Introduction

Reagents

Equipment

Procedure

Troubleshooting

Time Taken

Anticipated Results

References

Acknowledgements

Additional Declarations

Supplementary Files

Associated Publications

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Version 1

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Terms of Service

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