How to Detect Small Peptides by SDS-PAGE?

Does your sample contain proteins of interest that are <20 kDa? Please download a protocol on how to detect synthetic peptides using SDS-PAGE.

Shown is the resolution of cyanogen bromide fragments of myoglobin by (a) Tricine–SDS-PAGE and (b) Laemmli–SDS-PAGE using 10% T, 3% C gels.

Tricine-SDS-PAGE is commonly used to separate proteins in the mass range of 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa.

It is indeed very difficult to see the small peptide by SDS-PAGE. Tris-tricine gel will give you a better resolution. If you just want to detect the peptide, Mass Spec is still the best way to confirm the peptide identity.

Small peptide binds less Coomassie brilliant blue than larger protein. Thus smaller peptides are harder to detect by coomassie staining or silver staining. If you really want to see your peptide on the gel, you can try to load more samples. Changing the gel percentage won't help much unless you think your peptide migrated out of the gel. You can increase the percentage of cross linker in the regular 17% gel. In addition increase the pH of your resolving gel to 9.5 as compared to your regular 8.8. Plus, the addition of urea (4-8M) helps sharpen bands.

If you are going to use western, which is a way more sensitive detection method, please use Western instead of the gel staining. However the peptide may simply pass through the membrane. If you repeat the experiment, try to put two pieces of membrane and shorter time of transfer (less than 1 hour at 200 mA). 0.2um pore could be enough. You can get smaller pore but that shouldn't be necessary. You may want to try semi-dry transfer for 15-20 minutes at the recommended current density (mA/cm2) for the apparatus. A short 15 min transfer time works for most of the small peptides.

If you can plan ahead and synthesize a control small peptide labeled with biotin, you can monitor the transfer process and its ability to bind the membrane with streptavidin-conjugated HRP.

Please download this protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue staining, silver staining, and electroblotting.

Download the Protocol

• To download the protocol, click the download button below.

                       

---

How to calculate the peptide concentration?

Peptide content is not an indication of peptide purity; these are two measurements. Purity is determined by HPLC and indicates the presence/absence of contaminating peptides with undesired sequences. Net peptide content only gives information on the percent of total peptide versus total non-peptide components independently of the presence of multiple peptides. Net peptide content is accurately found by performing amino acid analysis or UV spectrophotometry.

It is difficult to determine the actual peptide concentration based on the weight of the lyophilized peptide. Lyophilized peptides may contain 10-70% water and salts by weight. More hydrophilic peptides generally contain more bound water and salts compared to hydrophobic peptides.

If the peptide has a chromophore in the sequence (W or Y residues), peptide concentration can be conveniently determined based on the extinction coefficient of these residues.

The following steps can be used for the calculations:

1. Molar extinction coefficients of chromophoric residues at 280 nm at neutral pH using a 1-cm cell:
   • Tryptophan 5560 AU/mmole/ml
   • Tyrosine 1200 AU/mmole/ml
2. The overall molar extinction coefficient of the peptide depends on the types and number of these choromophoric residues in the sequence.
3. Calculations: mg peptide per ml = (A280 x DF x MW) / e, where A280 is the actual absorbance of the solution at 280 nm in a 1-cm cell, DF is the dilution factor, MW is the molecular weight of the peptide and e is the molar extinction coefficient of each chromophore at 280 nm
4. Hypothetical example: A 50X diluted solution of a peptide with the sequence GRKKRRQRRRPPQQWDCDLYRPYEKT (MW = 3418) reads 0.5 AU at 280 nm in a 1-cm cell. To calculate the original peptide concentration in the stock peptide solution:
   mg peptide/ml = (0.5AU x 50 x 3418 mg/mmole) / [(1 x 5560) + (2 x 1200)] AU/mmole/ml = 10.7
5. Cautions:

• Any absorbance calculation assumes that the peptide is unfolded and the chromophores are exposed, which is usually the case in short, soluble peptides. If there are doubts about the solubility or the folding of the peptide, it is advisable to make the measurement under denaturing conditions (e.g., 6M GdnHCl or 8M urea). Obviously, these peptide solutions will be rendered useless, unless the denaturants are removed.
• If the sequence does not have Trp or Tyr, the only practical option is to do amino acid analysis.