How do I optimize electroblotting conditions onto PVDF membrane for N-terminal Edman sequencing? Our 12 kDa protein separates well on 1D SDS PAGE but does not appear on the PVDF blot for N-terminal sequencing. We are using the NuPAGE Transfer buffer with 20% MeOH from Invitrogen. What can we do to optimize the blotting conditions?

Answer: 

The blotting buffer generally works well for most proteins. However, some proteins may show poor electroblotting efficiency, and the choice of PVDF membrane, blotting buffer and blotting conditions should be optimized. During optimization it is an advantage to stain the gel after blotting and to use 2 layers of PVDF. Some large proteins (above 80 kDa) may be difficult to get out of the gel, and it can help to add 0.1% SDS to the buffer since SDS increases the mobility of the proteins. The same effect can be obtained by omitting MeOH from the buffer, because MeOH strips SDS from the protein.

Some small proteins (below 15 kDa) may move too quickly out of the gel and through to the first PVDF membrane. In that case, SDS should not be used and the MeOH concentration increased to 20%. Also the gel can be pre-soaked in blotting buffer for 5-10 mins before blotting. Choice of blotting buffers with a neutral pH (Tris-Glycine buffers), may be useful for very basic proteins with high isoelectric points. Basic proteins may be positively charged and the PVDF membrane should be placed on the other or on both sides of the gel. Glycine-containing buffers will give high glycine yield in the first Edman cycle, and the PVDF membrane should be washed extensively after staining. Use this PVDF transfer protocol for N-terminal Edman Sequencing

N-terminal sequencing is a requirement according to the ICH Q6B Guideline for characterization of recombinant proteins for clinical testing, and to demonstrate comparability and consistency between cGMP batches.

It is estimated that approximately half of all proteins cannot be directly sequenced by Edman degradation because they have a blocked N-terminal residue. No sequence data will be obtained if the N-terminus is blocked either naturally or as a result of sample processing. One problem frequently encountered is that the N-terminal residue is modified in such a way that it does not react with the Edman reagent phenyl isothiocyanate. For example, the blocked N-terminal residue may be an N-acetyl amino acid, a glycosylated amino acid, or a pyrrolidone carboxylate group. Of these, proteins with an acetylated amino acid are encountered most frequently. Evidence has been presented that about 80% of the soluble proteins in mammalian cells have acetylated N-terminal amino acids.

Examples of the N-terminal amino group of a polypeptide being modified covalently:

• acetylation − C( = O) − CH₃ : The positive charge on the N-terminal amino group may be eliminated by changing it to an acetyl group
• formylation − C( = O)H : The N-terminal methionine usually found after translation has an N-terminus blocked with a formyl group.
• Pyroglutamate:  An N-terminal glutamine can attack itself, forming a cyclic pyroglutamate group.

N-terminal block can also occur after isolation during sample manipulation. These following steps can help prevent this. Allow the gel to polymerize well since the free acrylamide might alkylate some amino acids, use a reducing agent in the electrophoresis running buffer (either sodium thioglycolate (0.1 to 1 mM) or 10 mM reduced glutathione) and avoid addition of acetic acid in your staining buffer.

Learn more about N-terminal Sequencing here and Sequencing Blocked N-terminals here .

Structural characterization of purified natural and recombinant proteins traditionally includes N-terminal Edman sequencing to confirm 5-15 amino acid residues from the amino terminus. N-terminal sequencing is a requirement according to the ICH Q6B Guideline for characterization of recombinant proteins for clinical testing, and to demonstrate comparability and consistency between cGMP batches. N-terminal Edman sequencing does not work for N-terminally blocked proteins, and the analysis is time-consuming with a cycle time of 40 minutes per amino acid. An alternative technique, ISD MALDI MS has been developed and demonstrated in several publications for top-down sequencing of intact peptides and proteins. 

MALDI ISD offers several key benefits compared to Edman sequencing:

• Both N- and C-terminal sequences of 20-50 residues can be obtained.
• N-terminally modified and blocked proteins (acetylated, pyroglutamate, PEGylated) can be sequenced.
• Data acquisition is fast.
• Very long sequence reads can be obtained with up to 80 residues from one single ISD MALDI mass spectrum.

MALDI ISD analysis can thus confirm expression and purification of the full length protein sequence, and detect unexpected truncations and modifications of the termini. The MALDI ISD analysis can also be applied to N-terminally blocked and PEGylated proteins. Read more about MALDI ISD here

For N-terminal Edman sequencing SDS PAGE separated proteins can be electroblotted onto PVDF membranes, the membranes stained and protein bands/spots cut out for N-terminal Edman sequencing. Compatible PVDF staining methods include Ponceau S, Coomassie Blue, Amido Black, and fluorescence Sypro Ruby staining. Silver staining and Western blots cannot be used in combination with Edman sequencing! You can download our protocols for electroblotting and PVDF staining below.

Pick ‘n Post PVDF Blotting and Staining Protocols

Hela den här kvalserien är som upplagd för misslyckande. Jag tittar på startelvan och det känns som att det är 1997 igen. Det är Johanssons, 100-åriga innebandyspelare, en mittback med samma spelförståelse som Jean Alain Boumsong, en impotent forward, en målvakt som trots sin ålder verkar har sett sina bästa dagar och så vidare. I don’t like it. Grundtryggheten finns där visst. Den svenska styrkan, samspelet, det defensiva jobbet, slitet. Men inte på samma sätt som tidigare. En frisk Edman saknas. Andreas Jakobsson, Michael Svensson eller Bjärred saknas där bak. En Andreas Isaksson anno 2004 fattas. Zlatan är avstängd. Ljungberg spelar i FC Fanchise. Jaja, oavgjort vore guld värt. En seger så otänkbart fantastisk att jag inte tänker på det.

Men.

2-0 till hemmalaget. Lite briljans från C7 och den här matchen, och kvalet, är över. Allt annat vore en sensation.

Nu drar det igång. 90 minuter obehag. Att man aldrig lär sig..

Erik Edman uttagen i VM-kvaltruppen mot Portugal. Här snackar vi om en spelare som inte har lirat en A-lagsmatch på ett drygt år, som var helt osynlig i matchen mot Österrike senast och som ärligt talat inte har gjort en bra landskamp sedan EM 2004. Han har knappt varit över halva planen heller sedan dess, i alla fall inte i landslagströjan…

Fullkomligt bedrövligt att man tas ut i en landslagstrupp på andra meriter än de rent sportsliga i ett svensk landslag år 2009! Kan förstå vitsen med att ha med en “pappa” eller en “kul kille” i en 23-mannatrupp under ett långt mästerskap, men inte i en enstaka match där den mesta av uppladdningen också sker på hemmaplan… 

Vissa är dock mer jämlika än andra i Lars-Rolands värld och det underliga är väl att jag fortfarande låter mig förvånas/uppröras över att de båda herrarna fortsätter att ägna sig åt den gamla socialistiska paradgrenen nepotism…

Avgå, Lagerbäck!