Home Technique / Proteomics and protein biochemistry / Protein expression, protein extraction and protein purification

Baculovirus expression system (10)

E.coli expression system (11)


Mammalian TAP-tagging technique - general protocols recommended protocol
Two step Tandem Affinity Purification (TAP) system to purify protein suitable for Mass Spectrometry analysis. (By Anne-Claude Ginras, 2003). ...

Recombinant protein expression and purification flowchart new hot recommended protocol
The Flowchart will help you to choose: * the right domain * the right expression system * how to optimize the expression levels of the target protein * the right method for protein extraction and clarification * the purification scheme * the corre ...

Preparation of Glycerol Stocks and Thawing of E.Coli Stocks protocol
The BL21 cells are fairly fussy in that, unlike other E. coli strains, their plasmids become unstable if the cells are stored in glycerol concentrations greater than 10%. For this reason, the cells should be stored in 8% glycerol and frozen quickly in liquid n ...

IPTG Induction of recombinant protein production new protocol
IPTG induction and bacterial growth A introduction and experimental protocol on BL21 e.coli recombinant protein expression and induction with IPTG. (Structural biology Prote ...

TCA protein precipitation protocol new protocol
From Bjorkerman lab at CIT. ...

Protocol for the Purification of Wnt Proteins protocol
Protocol by Karl Willert in Nusse Lab at Stanford University Medical School. ...

Removal of Nucleic Acids from Protein protocol
Methods using Polyethyleneimine (PEI) precipitation and Streptomycin sulfate precipitation. By Dr. Mario Lebendiker at The Hebrew University of Jerusalem. ...

Recombinant protein characterization by analytical ultracentrifugation protocol
With Analytical Ultracentifugation (AUC) the following protein characteristics can be determined: * Native Molecular Mass * Stoichiometry * Assembly Models * Conformation & Shape * Diffusion & Sedimentation * Assiociation Protocol from EMBL He ...

Storage of purified recombinant protein protocol
The method of choice depends completely on the stability of the protein and on when and how you want to use it later on. In general it is important to avoid storage conditions that are close to the stability limits of the protein (e.g. extreme pH or pH values ...

Protein extraction from whole tissues for isoelectric focusing new protocol
Protocol from University of Missouri - Columbia Proteomics Center. 1. SDS extraction followed by acetone precipitation. 2. Phenol extraction followed by methanolic ammonium acetate precipitation. ...

Enhancing solubility during protein expression in e.coli new review
A tech note from Novagen on protein solubility in e.coli expression system. The choice of vector and expression host can significantly increase the activity and amount of target protein present in the soluble fraction. Some suggestions are given in this tech n ...

Detergent used for protein biochemistry and proteomics study review
Properties of Detergents Table by Dr. Shaun D. Black (University of Texas Health Center at Tyler). List of Purity, MW (monomer), CMC (mM), CMC Conditions, Aggregation, MW (micelle) of ionic, non-ionic and Zwitterionic Detergents. Detergent molecule has ...

Purification Tricks for Recombinant Proteins new review
An introduction of recombinant protein purification and application tricks by Bill Campbell at Michigan Tech. Recombinant proteins are made from cloned DNA sequences which usually encode an enzyme or protein with known function. Special vectors (a vector is ...

Protease inhibitors new review
Summary: Protease inhibitors used in protein extraction step. Content: Protease inhibitors essential for prevent protein extracts from proteolysis, dephosphorylation, and other protein degradation/modification related activities. Commonly used protease i ...

Protein purification protocols and resources new recommended site
A huge collection of protocols on protein purification by Dr. Mario Lebendiker at The Hebrew University of Jerusalem. ...

Dye-binding protein assay giving very low signals troubleshooting
I've eluted some polyclonal antibodies from an antigen column, and dialysed them into a suitable buffer - the strange thing is that although the absorbance at 280nm indicates that the proteins are at a certain, relatively high level (e.g. 1mg/ml), whe ...

Question about Protein A purification of IgG troubleshooting
1. What is the molecular basis for the selective binding of IgG by protein A? 2. What is the difference between Protein A and Protein G in relation to their Ig class binding specificities? ... ... ...

protein precipitation troubleshooting
I am looking for a protein precipitation method for very low concentrated samples (50ug/ml) I have tried chloroform methanol but that doesn't work. My samples are in phosphate buffer + n-octylglucoside. ... ... ...

Bacterial protein expression and purification discussion troubleshooting
I am working with the pBAD bacterial his tag expression system from Invitrogen, and am trying to purify the his-tag protein. I have checked a number of methods to lyse the cells to release the protein, but in each case the vast majority of my protein ...

His-tags and DNA-binding troubleshooting
Has anyone come across a situation where a 6XHis tag has affected the DNA-binding of a DNA-binding protein? (the pH is 8 so unlikely to be +ve charge). I know the folding could be affected. Just wondered if anyone had seen such a phenomenon... ...

Troubleshooting affinity chromatography new troubleshooting
Affinity chromatography purification troubleshooting guide from Roche. Problems: 1. Tagged protein appears in washes, does not bind to affinity resin. 2. Tagged protein remains bound to column, does not elute in Elution Buffer. 3. Extra proteins in elua ...

His-tag protein purification new troubleshooting
Use of His-tag to coprecipitate a GPCR with protein partners 14.10.2003 10:18 I am working on GPCR-partners interactions, especially on PDZ type interactions. I detected specific interactions by chromatography affinity using a peptide mimicking the C-ter ...

Graphical codon usage analyser for gene expression experiments new software
Differences in codon usage among organisms can lead to a variety of problems concerning heterologous gene expression. The gcua tools display the codon bias in a graphical manner. Reference: Monitoring dynamic expression of nuclear genes in Chlamydomonas ...

More protein expression protocols

Production of recombinant proteins in mammalian cells.
(Curr Protoc Protein Sci. 2001 May;Chapter 5:Unit5.10.)
Chen S, Gray D, Ma J, Subramanian S.
Chiron Corporation, Emeryville, California, USA.

The best strategy for consistent production of larger quantities of pure protein is stable expression. Popular hosts for stable expression are Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK-21) cells, myeloma cells, and the transformed kidney cell line 293. Protocols for stable production in CHO cells are described in this unit. Typical methods for transfection using commercially available plasmid expression vectors are described, along with methods to select for stable expression and methods for amplifying the expression level in the transfected cell. Following this, procedures are presented for efficient cell growth to obtain significant amounts of protein product. Support protocols describe freezing of cells, determination of growth rates, determination of specific productivity of cells, preparing samples for assay, and setting up a 10-day shaker-flask growth curve.


(Protein expression reviews)
1: Forstner M, Leder L, Mayr LM.
Optimization of protein expression systems for modern drug discovery.
Expert Rev Proteomics. 2007 Feb;4(1):67-78.

2: Hartley JL.
Cloning technologies for protein expression and purification.
Curr Opin Biotechnol. 2006 Aug;17(4):359-66. Epub 2006 Jul 12.

3: Barnes LM, Dickson AJ.
Mammalian cell factories for efficient and stable protein expression.
Curr Opin Biotechnol. 2006 Aug;17(4):381-6. Epub 2006 Jun 27.

4: Esposito D, Chatterjee DK.
Enhancement of soluble protein expression through the use of fusion tags.
Curr Opin Biotechnol. 2006 Aug;17(4):353-8. Epub 2006 Jun 15.

5: Cox B, Kislinger T, Emili A.
Integrating gene and protein expression data: pattern analysis and profile
mining.
Methods. 2005 Mar;35(3):303-14. Epub 2005 Jan 12.

6: Hunt I.
From gene to protein: a review of new and enabling technologies for
multi-parallel protein expression.
Protein Expr Purif. 2005 Mar;40(1):1-22.

7: Cabrita LD, Bottomley SP.
Protein expression and refolding--a practical guide to getting the most out of
inclusion bodies.
Biotechnol Annu Rev. 2004;10:31-50.

8: Greene JJ.
Host cell compatibility in protein expression.
Methods Mol Biol. 2004;267:3-14.

9: Freeman WM, Hemby SE.
Proteomics for protein expression profiling in neuroscience.
Neurochem Res. 2004 Jun;29(6):1065-81.

10: Pearlberg J, LaBaer J.
Protein expression clone repositories for functional proteomics.
Curr Opin Chem Biol. 2004 Feb;8(1):98-102.

11: Yokoyama S.
Protein expression systems for structural genomics and proteomics.
Curr Opin Chem Biol. 2003 Feb;7(1):39-43.

12: Chambers SP.
High-throughput protein expression for the post-genomic era.
Drug Discov Today. 2002 Jul 15;7(14):759-65.

13: Andersen DC, Krummen L.
Recombinant protein expression for therapeutic applications.
Curr Opin Biotechnol. 2002 Apr;13(2):117-23.

14: Lesley SA.
High-throughput proteomics: protein expression and purification in the
postgenomic world.
Protein Expr Purif. 2001 Jul;22(2):159-64.

15: Heifetz PB, Tuttle AM.
Protein expression in plastids.
Curr Opin Plant Biol. 2001 Apr;4(2):157-61.

16: Konz JO, King J, Cooney CL.
Effects of oxygen on recombinant protein expression.
Biotechnol Prog. 1998 May-Jun;14(3):393-409.

17: Mastrangelo AJ, Betenbaugh MJ.
Overcoming apoptosis: new methods for improving protein-expression systems.
Trends Biotechnol. 1998 Feb;16(2):88-95.

18: Baker RT.
Protein expression using ubiquitin fusion and cleavage.
Curr Opin Biotechnol. 1996 Oct;7(5):541-6.

19: Kurland C, Gallant J.
Errors of heterologous protein expression.
Curr Opin Biotechnol. 1996 Oct;7(5):489-93.

20: Cole PA.
Chaperone-assisted protein expression.
Structure. 1996 Mar 15;4(3):239-42. No abstract available.


(Protein purification reviews)
1: Roy I, Mondal K, Gupta MN.
Leveraging protein purification strategies in proteomics.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Nov 30; [Epub ahead of
print]

2: Franzreb M, Siemann-Herzberg M, Hobley TJ, Thomas OR.
Protein purification using magnetic adsorbent particles.
Appl Microbiol Biotechnol. 2006 May;70(5):505-16. Epub 2006 Feb 23.

3: Banki MR, Wood DW.
Inteins and affinity resin substitutes for protein purification and scale up.
Microb Cell Fact. 2005 Nov 11;4:32.

4: Linhult M, Gulich S, Hober S.
Affinity ligands for industrial protein purification.
Protein Pept Lett. 2005 May;12(4):305-10.

5: Hilbrig F, Freitag R.
Protein purification by affinity precipitation.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jun 25;790(1-2):79-90.


6: Anspach FB, Curbelo D, Hartmann R, Garke G, Deckwer WD.
Expanded-bed chromatography in primary protein purification.
J Chromatogr A. 1999 Dec 31;865(1-2):129-44.

7: Galaev IY.
New methods of protein purification. Affinity ultrafiltration.
Biochemistry (Mosc). 1999 Aug;64(8):849-56.

8: Galaev IY.
New methods of protein purification. Displacement chromatography.
Biochemistry (Mosc). 1998 Nov;63(11):1258-65.

9: Galaev IY.
New methods of protein purification. Expanded bed chromatography.
Biochemistry (Mosc). 1998 Jun;63(6):619-24.

10: Irwin JA, Tipton KF.
Affinity precipitation: a novel approach to protein purification.
Essays Biochem. 1995;29:137-56.

11: Gatlin LA, Nail SL.
Protein purification process engineering. Freeze drying: A practical overview.
Bioprocess Technol. 1994;18:317-67.

Protein expression technique books
(Biowww bookshelf)
Protein expression technique books

Protein purification technique books
(Biowww bookshelf)
Benchtop books on protein purification.

Plasmid Expression Vectors Carrying the T7 Late Promoter
(Molecular Cell Biology)
7. Recombinant DNA and Genomics

Engineered Protein Expression
(Cancer Medicine)
Part II Scientific Foundation, Section 1: Cancer Biology 2. Molecular Biology, Genomics, and Proteomics

Protein expression in e.coli problem
(Forum)
Suggestion of codon optimization

protein purification and antimicrobial assay
(Forum)
problem elution and purification of proteins

protein precipitation after IMAC
(Forum)
35kDa protein with His-tag N-terminal precipitation

protein concentration
(Forum)
Protein concentration measurement

HisTag Protein purification WITHOUT imidazol
(Forum)
purify a his-tag protein

Endogenous protein and overexpressed protein detection
(Forum)
Troubleshooting undetectable endogenous protein

protein elution from gel
(Forum)
elute proteins from electrophoretic gel either SDS-PAGE or native

Myofibrillar extraction from mouse heart
(Forum)
protocol for the extraction of myofibrillar proteins from atria and ventricles of mouse heart

Protein overexpression with IPTG
(Forum)
problem to get a kinase domain overexpressed with 0,5 mM IPTG for 2h at 37 C in supernatant.

GST purification probelms
(Forum)
GST fusion protein expression and purification troubleshooting

problem in cleavage of fusion protein
(Forum)
cleavage of maltose-binding protein

Purification of enzymatically active protein from PAGE
(Forum)
purify the digested product of His-tagged fusion proteins pET System


Protein solubility
(Forum)
E.coli expression protein solubility problem

Protein refolding problems
(Forum)
refold a purified cysteine protease via dialyis of 6M urea

How to make the soluble protein from the insoluble protein
(Forum)
how to purify the soluble protein from the insoluble protein


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