Are you trying to find a way to detect small peptides clearly and sensitively, using SDS-PAGE and Western blotting?

If your sample contains proteins of interest that are less than 20 kDa, you can download a protocol that explains how to detect synthetic peptides or small molecular weight proteins using SDS-PAGE/Western blotting.

Here are some tips to help ensure your Western performs optimally.

• Use a PVDF membrane, not an NC membrane.
• Choose a pore size of 0.2 microns, not 0.45 microns.
• Use a shorter transfer time (such as 1 hour for wet transfer), or consider a semi-dry or vacuum-assisted detection method.
• Select gradient PAGE of 8-15% or 4-20%.
• If you're using a conventional Western blotting method with shaking, consider fixing the blot membrane with aldehyde, formaldehyde, or paraformaldehyde before applying it. Note that such fixation may affect the peptide immunoreactivity due to chemical modification.
• Consider using the Tricine PAGE, which is specifically designed for small and/or hydrophobic proteins.

If you want to validate your antibodies, here are some useful tips:

• Optimize your antibody protocols for each application. Make sure to optimize your protocols and antibody dilutions for each new application. Here are a few useful tips.
• Test the specificity, sensitivity, and reproducibility of the antibodies. Run the experiments three times and compare the antibodies between lots or different samples.
• Use both positive and negative controls for each experiment.
• Store the antibodies accordingly. Since antibodies have limited shelf lives, do not store working dilutions for later use.

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 a commonly used method for separating proteins in the mass range of 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa.

However, it can be difficult to detect small peptides using this method. In this case, a Tris-tricine gel may provide better resolution. If you only need to detect the peptide, Mass Spec is the best way to confirm its identity.

Smaller peptides bind less Coomassie brilliant blue than larger proteins, which makes them harder to detect by Coomassie staining or silver staining. If you want to see your peptide on the gel, you can try loading more samples. However, changing the gel percentage won't help much, unless you think your peptide migrated out of the gel. Instead, you can increase the percentage of the crosslinker in the regular 17% gel. Also, raising the pH of your resolving gel to 9.5 as compared to your regular 8.8, and adding urea (4-8M) can help sharpen bands.

If you plan to use a Western blot, which is a more sensitive detection method, it is better to use Western instead of gel staining. However, it can still be challenging to detect small peptides efficiently by the conventional Western blotting method because the peptides readily detach from the blotted membrane. To improve the detection, you can try using two pieces of membrane, a shorter time of transfer (less than 1 hour at 200 mA), and a 0.2um pore. Semi-dry transfer for 15-20 minutes at the recommended current density (mA/cm2) for the apparatus may also work for most of the small peptides.

Another method that can be used to shorten the time required for immunodetection steps is vacuum-assisted detection. Please check this reference: A new approach to detect small peptides clearly and sensitively by Western blotting using a vacuum-assisted detection method.

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.

For more information on Tricine-SDS-PAGE, including efficient methods for Coomassie blue staining, silver staining, and electroblotting, please download this protocol.

Download the Protocol

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

                       

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How to calculate the peptide concentration?

Calculating peptide concentration is not the same as determining peptide purity. Purity is measured by HPLC and indicates the presence or absence of contaminants with undesired sequences. On the other hand, peptide content only gives information on the percentage of total peptide versus total non-peptide components independently of the presence of multiple peptides.

Net peptide content can be accurately determined 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, with more hydrophilic peptides generally containing more bound water and salts compared to hydrophobic peptides.

If the peptide contains tryptophan (W) or tyrosine (Y) residues, its concentration can be conveniently determined based on the extinction coefficient of these residues. The molar extinction coefficients of chromophoric residues at 280 nm at neutral pH using a 1-cm cell are: tryptophan 5560 AU/mmole/ml and tyrosine 1200 AU/mmole/ml. The overall molar extinction coefficient of the peptide depends on the types and number of these choromophoric residues in the sequence.

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:

• It's important to note that 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 tryptophan or tyrosine, the only practical option is to do amino acid analysis.