Enzymatic and Chemical Deblocking of N-Terminally Modified Proteins
307
Table 22.2. Extraction of a-lactalbumin from PVDF membranes witb different volatile solutions
Immobilon-P
Fluorotrans
0.1 % TFA, 40 % acetonitrile
0.1 M ammonium bicarbonate, 40 % acetonitrile
+
++
+
++
+++
+++
++
+
++
+
++
+++
+++
+++
0.1 M ammonium bicarbonate, 40 % acetonitrile, 2 % ~- ME
30 % acetic acid
40 % acetic acid
70 % acetic acid
90 % acetic acid
70 % acetic acid, 30 % acetonitrile
Metbods: A vial oflow molecular marker proteins (Pharmacia Biotec) was dissolved in 200 III of SDS
sample buffer. 5 III of tbe protein solution was applied to SDS-PAGE and tbe separated proteins were
electroblotted onto a PVDF membrane, staining witb Coomassie Blue. Three blots for a-lactalbumin
were used to elute the proteins out of tbe wet membrane with different extraction solutions at room
temperature for 1 h with sonication. The relative recovery of proteins was measured: +++, >50 %,
++, >30-50 %, +, > 10-30 %, -,none.
Table 22.3. Extraction yields
of proteins from different type
of membranes
a-Immobilon-P
Hyperbond
Immobilon-CD
Fluorotrans
ProBiott
Transblot
Polypropylene
Teflon tape
Teflon (GoreTex)
Carbonic anhydrase
+++
++
+
+
++
++
+
+++
++
a-lactalbumin
+++
+
+
+++
+++
+++
+++
Methods: A vial oflow molecular marker proteins (Pharmacia Biotech) was dissolved in 200 III of SDS sample buffer. 5 III of the protein solution was applied to SDS-PAGE and tbe separated alactalbumin and carbonic anhydrase were electroblotted onto different PVD F membranes. The proteins were extracted with 70 %
acetic acid at room temperature for 1 h with sonication. The relative recovery of proteins was measured: +++. >50 %, ++,
>30-50%, +, >10-30%, -none.
ethanol or 70 % acetic acid in 30 % acetonitrile. It is recommended to use the
Immobilon-P membrane or teflon membrane (GoreTex) for more efficient elution than Immobilon-PSQ, -CD, Hyperbond, Fluorotrans, Problott, Transblott
and polypropylene membranes (Table 22.3, Fig. 3). Fig. 2 shows amino acid
sequence of deblocked super oxide dis mutase (SOD) eluted from Immobilon-P
membrane using 70 % acetic acid by sonication for lh. The condition for deblocking are the same as for neurotensin, except that for deblocking acetylatedDAP was used. For this reason only sequence of SOD after cleavage with DAP
(GPVQGTI..) was analysed and not additionally the sequence of DAP, as
described for deblocking of neurotensin. The interpretation of data obtained in
this way is much easier.
Summarizing the results, DAP can be used for the cleavage of acetylated peptides and proteins. Although yields may still vary from 1-50 %, depending on the
307
Table 22.2. Extraction of a-lactalbumin from PVDF membranes witb different volatile solutions
Immobilon-P
Fluorotrans
0.1 % TFA, 40 % acetonitrile
0.1 M ammonium bicarbonate, 40 % acetonitrile
+
++
+
++
+++
+++
++
+
++
+
++
+++
+++
+++
0.1 M ammonium bicarbonate, 40 % acetonitrile, 2 % ~- ME
30 % acetic acid
40 % acetic acid
70 % acetic acid
90 % acetic acid
70 % acetic acid, 30 % acetonitrile
Metbods: A vial oflow molecular marker proteins (Pharmacia Biotec) was dissolved in 200 III of SDS
sample buffer. 5 III of tbe protein solution was applied to SDS-PAGE and tbe separated proteins were
electroblotted onto a PVDF membrane, staining witb Coomassie Blue. Three blots for a-lactalbumin
were used to elute the proteins out of tbe wet membrane with different extraction solutions at room
temperature for 1 h with sonication. The relative recovery of proteins was measured: +++, >50 %,
++, >30-50 %, +, > 10-30 %, -,none.
Table 22.3. Extraction yields
of proteins from different type
of membranes
a-Immobilon-P
Hyperbond
Immobilon-CD
Fluorotrans
ProBiott
Transblot
Polypropylene
Teflon tape
Teflon (GoreTex)
Carbonic anhydrase
+++
++
+
+
++
++
+
+++
++
a-lactalbumin
+++
+
+
+++
+++
+++
+++
Methods: A vial oflow molecular marker proteins (Pharmacia Biotech) was dissolved in 200 III of SDS sample buffer. 5 III of the protein solution was applied to SDS-PAGE and tbe separated alactalbumin and carbonic anhydrase were electroblotted onto different PVD F membranes. The proteins were extracted with 70 %
acetic acid at room temperature for 1 h with sonication. The relative recovery of proteins was measured: +++. >50 %, ++,
>30-50%, +, >10-30%, -none.
ethanol or 70 % acetic acid in 30 % acetonitrile. It is recommended to use the
Immobilon-P membrane or teflon membrane (GoreTex) for more efficient elution than Immobilon-PSQ, -CD, Hyperbond, Fluorotrans, Problott, Transblott
and polypropylene membranes (Table 22.3, Fig. 3). Fig. 2 shows amino acid
sequence of deblocked super oxide dis mutase (SOD) eluted from Immobilon-P
membrane using 70 % acetic acid by sonication for lh. The condition for deblocking are the same as for neurotensin, except that for deblocking acetylatedDAP was used. For this reason only sequence of SOD after cleavage with DAP
(GPVQGTI..) was analysed and not additionally the sequence of DAP, as
described for deblocking of neurotensin. The interpretation of data obtained in
this way is much easier.
Summarizing the results, DAP can be used for the cleavage of acetylated peptides and proteins. Although yields may still vary from 1-50 %, depending on the