1.3.7 Sample Losses
Sample loss of small amounts of protein is often caused by adsorption to purification apparatus. To avoid this problem try eliminating as many handling steps as possible.
A. Avoid dialysis by applying sample from gel filtration or ion exchange chromatography directly to a reverse phase HPLC separation set up.
B. Use polyethylene or polypropylene micro centrifuge tubes keeping protein mass to plastic area as high as possible.
C. Avoid drying or lyophilizing samples completely.
1.3.8 Tips for SDS Gel Electrophoresis Prior to Sequencing
A. Use ultra-pure reagents.
B. Make up stock acrylamide solution fresh every 2 to 3 weeks.
C. De-ionize the acrylamide stock solution with ion exchange resin and store it in a brown glass container in a cool place.
D. Polymerize gel overnight to minimize any amino-reactive free acrylamide.
E. Pre-run the gel (5-10 minutes) before loading your sample.
F. Denature sample for 0.5-1hr at 65oC - avoid boiling sample.
1.3.9 PVDF Types for Transferring Sequencing Samples from SDS Gels
A. Weak binding PVDF-suitable for proteins from 5kD up to <100kD
Immobilon-P membranes/Millipore Corp.
B. Strong binders-more suitable for small polypeptides (<5kD)
Perkin Elmer/Applied Biosystems' "ProBlott"
Millipore's "Immobilon P (SQ)"
C. Nitrocellulose is not compatible with the Edman chemistry.
1.3.10 Details for Sample Shipping to Creative Proteomics
A. Please, fill the Edman protein sequencing form with information about the samples (e.g. sample name, sample type, MW, protein amount, staining method) and the requested number of N-terminal sequencing steps. Sign the form!
B. Send us an email before shipping the samples
C. The samples should be in an Eppendorf reaction tube sealed by Parafilm. Protein samples on PVDF membrane can either be shipped as small and dry membrane slides in reaction tubes or as a dried PVDF membrane in plastic foil with enclosed description of the protein bands which should be analyzed. We cut the marked protein band for analysis.
D. Liquid samples should be sent in frozen state.
E. Lyophilized sample and samples blotted on PVDF membrane can typically shipped at room temperature.
F. Please, send your samples in a padded envelope or in a box together with the Edman protein sequencing form.
1.4 Our Recommendation for Protein Blotting
1.4.1 Semidry PVDF Blotting Protocol
We recommend the following protocol for semidry blotting on PVDF membrane:
- PVDF membrane: Immobilon P membrane or comparable
- Blot buffer: 50mM sodium borate, pH 9.0 / 20% methanol (HPLC quality)
(0.1% SDS can be added to blotting buffer if protein above 40kDa should be sequenced)
- Blotting condition: 1mA/cm1 PVDF membrane for 2-3 hours at 4°C
1.4.2 Protein Staining of PVDF Membranes
Ponceau Red Staining
Staining solution (0.5% Ponceau S, 1% acetic acid in Milli-Q water):
0.25g Ponceau S
0.5mL acetic acid
Add 50mL Milli-Q water
1. Wash the PVDF blot membrane 2x 3 minutes with plenty Milli-Q water.
2. Stain the PVDF membrane with Ponceau S staining solution for 1-3 minutes.
3. Destain the PVDF blot membrane under visual control with Milli-Q water until protein bands are well visible.
4. Dry the PVDF membrane.
Staining solution (0.005% sulforhodamine, 0.2% acetic acid, 30% methanol in Milli-Q water):
1mL acetic acid
Add 500mL Milli-Q water
1. Wash the PVDF blot membrane 2x 10 minutes with plenty Milli-Q water.
2. Dry the PVDF membrane at room temperature!
3. Stain the PVDF membrane in Sulforhodamine staining solution for 1-2 minutes.
4. Wash the PVDF membrane with Milli-Q water shortly.
5. Dry the PVDF membrane.
CBB R250 Staining
Staining solution (0.1% CBB R250, 10% acetic acid, 40% methanol in Milli-Q water):
0.1g CBB R250
10mL acetic acid
Add 100mL in Milli-Q water
Destaining solution (10% acetic acid, 40% methanol in Milli-Q water):
10mL acetic acid
Add 100mL Milli-Q water
1. Stain the PVDF blot membrane for 5 minutes in CBB R250 staining solution
2. Destain the PVDF blot membrane for 3 x 5 minutes with destaining solution under visual control until protein bands are well visible.
3. Dry the PVDF membrane.
1.5 Application Field of N-Terminal Sequencing
Improvements in this field have allowed minute quantities of purified protein / peptides to be easily sequenced. Protein Sequencing can now be routinely used as an analytical tool in life science research
A. Determination the N-terminal amino acids of a protein
B. Determination the N-terminal amino acids of a peptide
C. Check of the correct translation of a recombinant protein
D. Check of the sequence and purity of a recombinant protein
E. Check of the sequence and purity of a synthetic peptide
F. Determination of complete protein sequence in combination with additional methods
G. Quality control N-terminal sequencing is most commonly used to identify unknown proteins, confirm protein identity and quality (often for quality control of recombinant proteins), and identify protein N-terminus and cleavage sites
H. Homology based protein identification.
We have extensive experiences in this technology and will work with you from any steps in the workflow. Please contact us for a free project consultation.