A Simple Protocol to Refold Peptides or Small Proteins

Understanding the process of peptide folding is a critical first step toward understanding protein folding. Depending on the temperature and solvent conditions, peptides are highly flexible and can adopt a variety of conformations in solution. Many unfolded peptides could spontaneously refold in vitro to form a native protein with full biological activity in the absence of other factors.

The primary sequence contains all the information to define the three dimensional structure of a protein and its biological functions. The mutation or deletion of any amino acid may have a big impact on folding and stability. It takes nanoseconds (ns) for the peptide to form an intermolecular contact. The timescales of loop closing is 10 nanosecond (ns). The formation of alpha-helical peptides is 200 ns,  beta hairpins and mini-proteins in 1–10 ms timescale. Many studies had a very good agreement between measured and calculated folding rates. Many factors such as temperature, pH,  molecular chaperones, salts, and denaturant may affect a peptide in reaching its native state.

So it is critical to minimize factors that affect protein refolding. A successful folding should have inadequate denaturant concentrations to destabilize the native state of a peptide or protein. GnHCL can be used in order to disrupt the hydrophobic interactions within the tertiary structure.

  1. The linear, purified peptide (1 mg) is dissolved in 6M GnHCl/300mM PBS (500µL) for about 1-6 hours before refolding studies are carried out.
  2. This solution is added SLOWLY drop by drop (3 min) to the degassed 50mMTris / 1mM EDTA pH~8 (3.9 mL) solution, containing reduced glutathione (GSH) (6 mg) and oxidized glutathione (GSSG) (2mg).
  3. The solution should be clear without precipitation.
  4. Add the solution into a dialysis tube with appropriate cutoff (MW 1K or 3K according to your peptide molecular weight).
  5. Exchange the dialysis buffer with decreasing concentration of GnHCL
    according to any of your favorite dialysis protocol. Use your own dialysis buffer according to your downstream experiments. (Dialysis buffer example: PBS, 2mM DTT)


  • Current Opinion in Structural Biology 2003, 13:168–174
  • Reversible Peptide Folding in Solution by Molecular Dynamics Simulation, J. Mol. Biol. (1998) 280, 925-932

LifeTein Peptides Used for Pulldown Assay

LifeTein synthesized multiple peptides for the pulldown experiments. The peptides were synthesized with biotin on the N terminus and an aminohexanoic acid linker. Two types of peptides were designed: peptide without acetylation and peptide with multiple lysines (up to five sites) were acetylated in the synthetic peptides.

These peptides were bound to streptavidin-agarose beads and used for pulldown experiments using cell lysates.  Peptides, streptavidin agarose beads, and cell lysates were permitted to bind for overnight at 4 °C,  then beads were pelleted, washed five times using spin columns, and proteins were eluted in sample buffer and analyzed by SDS-PAGE and immunoblotting.

All synthetic peptides had an N-terminal biotin. After incubation, peptides were spotted on nitrocellulose membranes and immunoblotted to detect acetylated Lysine (AcK) or total biotinylated peptide. Data is shown below.

Acetylated lysine peptide

Dot blot for biotin peptide with pulldown assay


Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-Responsive Intracellular Compartment, The Journal of Biological Chemistry, January 5, 2015, doi: 10.1074/jbc.M114.603977 jbc.M114.603977.


Synthetic Peptides Used for indirect ELISA


Peptides can be used for ELISA assay. A peptide-based indirect ELISA was used to screen a population of 40 Multiple sclerosis patients and 39 healthy controls. The encephalitogenic myelin oligodendrocyte glycoprotein (MOG)35–55 synthetic peptides were synthesized by LifeTein.

All MOG peptides or Mycobacterium avium subspecies paratuberculosis (MAP) peptides were synthesized at >90% purity by LifeTein to make sure the ELISA results are clean and consistent. The plates were coated with peptides. After overnight incubation, the plate was blocked, rinsed, and late react with antibodies according to the protocol. In silico analysis identified two peptides belonging to MAP and BCG, which share sequence homology with MOG(35–55). The peptide-based indirect ELISA data showed that sharing of highly conserved linear amino acidic sequences is necessary to elicit antibody-mediated cross-reactivity.

These findings concluded that the presence of MOG (35–55)-specific antibodies in multiple sclerosis pathogenesis. This can be used as a diagnostic biomarker in multiple sclerosis.

 [PDF] Evaluation of the humoral response against mycobacterial peptides, homologous to MOG35–55, in multiple sclerosis patients

MG Marrosu, LA Sechi – 2014

 All peptides were synthesized at N90% purity commercially (LifeTein, South
Plainfield, NJ 07080 US). Purified peptides were prepared as [10 mM] stock solutions,
and were stored in single- use aliquots at −80 °C. 2.3. ELISA 

LifeTein Peptide Cited in Cell

Peptide library is increasingly used to define antibody epitopes and substrate specificities of protein kinases. For epitope mapping, overlapping peptides are made to span the antigenic protein sequence. The antigenic determinant recognized by a monoclonal antibody can then be screened and defined. The alanine scanning method can also be used to assess that residue’s contribution to antibody binding and to determine which substitutions affect antibody recognition (mutational analysis). Unrelated synthetic peptides can be used to evaluate the antibody cross-reactivity.

Overlapping peptides from LifeTein were used to map the region of Fragment 3 by epitope mapping of anti-Fzd2 antibody. This anti-Fzd2 antibody was found to reduce tumor growth. Wnt signaling plays a critical role in colorectal cancer. Researchers found that Wnt receptor Frizzled2 (Fzd2) and its ligands Wnt5a/b are elevated in metastatic liver, lung, colon, and breast cancer cell lines. Their high level expression correlates with markers of epithelial-mesenchymal transition (EMT). By epitope mapping using synthetic peptides from LifeTein, the researchers mapped the epitope to a specific region. The antibody to Fzd2 was found to reduce cell migration and invasion. Targeting this pathway may provide a cure for patients with tumors expressing high amount of Fzd2 and Wnt5a/b.

We have developed an antibody to Fzd2 that reduces cell migration and invasion and inhibits tumor growth and metastasis in xenografts. We propose that targeting this pathway could provide benefit for patients with tumors expressing high levels of Fzd2 and Wnt5a/b.

Phospho-specific antibodies by LifeTein published in Nature

Jia Shen. et al. EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2. Nature 497, 383–387 (16 May 2013), doi:10.1038/nature12080 LifeTein helped designed and synthesized a series of phosphorylated s. Then the peptides were used for phospho-specific productions. The phospo-specific antibodies by LifeTein were confirmed to react with the epidermal growth factor receptor (EGFR). The Hung’s lab showed that AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells. … The following peptides were chemically synthesized for in mice (Lifetein Conc.), Elisa verification (LifeteinConc.) and peptide competition assay in immunohistochemistry (IHC)… Supplementary information

Synthesis of multiple antigenic peptides: strategies and limitations

Synthesis of multiple antigenic peptides: strategies and limitations

Dendrimeric platforms such as multiple antigenic peptides (MAP) can be synthesized either entirely by solid-phase methods (SPPS, direct approach) or by conjugation in SPPS-made building blocks (indirect approach).

MAP peptide synthesis

MAP peptide synthesis


SPPS is the preferred method by LifeTein. The synthesis approach requires a branched poly-lysine core. Each branch is elongated into the corresponding epitope by stepwise SPPS. The disadvantage of this approach is that the synthetic errors could happened and cause microheterogeneity in the final materials. However the cost is lower and less time-consuming than the indirect approach. For very long linear peptides, it is more advantageous to use the SPPS method.

The MAP synthesis may not always meet with success. The solubility of the peptide epitope can also become an issue and is difficult to predict for long epitopes. It is recommended to carefully design and analyze the linear epitope before MAP synthesis.

Studies showed that synthesis with Ahx linker in the lysine core had better isolated yield. It is possible that the flexibilizing effect of Ahx helps in keeping peptide chains properly solvated during synthesis, preventing aggregation and hence increasing the amount of viable growing peptide sequences.


The Structural Basis of Peptide-Protein Binding Strategies

peptide protein binding strategy

Peptide protein binding strategies: Structure, Volume 18, Issue 2, 188-199, 10 February 2010



  • After binding its partners, most peptides do not introduce any conformational changes
  • The interfaces of peptide-protein have more hydrogen bonds
  • The peptide hotspots are important for the binding
  • Peptides prefer to bind in the largest pockets on the protein surface

Read more from here.

LifeTein is pleased to offer a free, comprehensive web-based peptide analysis tool. This tool will allow your research team to overcome common difficulties inherent in protein analysis and peptide antigen design.


Peptide analysis tool-LifeTein

Amino acid composition of cell-penetrating peptides (CPPs)

Cell-penetrating peptides (CPPs) such as the HIV TAT peptides are able to enter cell by direct translocation and endocytosis. Click here to see details about the CPP: http://lifetein.com/Cell_Penetrating_Peptides.html

cell penetrating peptide entry mechanism

Cell Penetrating Peptides

The following table shows a selection of currently known CPPs, their origins and sequences.




Tat (48-60)

HIV-1 protein



Igl-1 homeodomain



(Stearyl = CH3(CH2)16CO-)


(X=6-aminohexanoic acid)


Obesity Treatment: A New Peptide Drug

Targeted Drug Melts Fat in Obese Monkeys

Currently, only two Food and Drug Administration (FDA) approved drugs for weight loss are available in the United States: the appetite suppressant phentermine and the inhibitor of fat absorption orlistat.

An MD Anderson group designed a new peptide drug: CKGGRAKDC-GG-D(KLAKLAK)2 (termed adipotide). This is a synthetic peptide that triggers cell death. These data showed that the peptide may be useful for treating obesity in humans.

Tthe MD Anderson group used a peptide library to screen and identify regions that bind to specific vascular cells. The interaction identified will be used as effective drugs to target particular protein functions.


This video explains factors that have contributed to the obesity epidemic.