2. RESULTS AND DISCUSSION
2.5.4 Dot Blot analysis of Lys63 linkage antibody with different Lys linked di-ubiquitin
Dot blot is a simple technique for detecting, analyzing, and identifying specific proteins, in which the samples are spotted through circular templates directly onto the surface of membrane. The method is based on the antigen-antibody recognition by using a first antibody against the membrane-immobilized antigen, and a second label-conjugated antibody for detection of the first antibody (Fig. 78).
HRP
Primary Antibody
Scondary Antibody
Protein Binding Membrane
Immobilized Protein Coupled Enzyme ECL Detection
Figure 78. Schematic representation of dot blot experiment. The proteins immobilized on the membrane are probed with a specific antibody and a matching specific detection reagent.
The immuno-detection of the two different Lys linked di-ubiquitin peptides to a specific ubiquitin antibody, namely the monoclonal K63 linkage polyubiquitin antibody (clone HWA4C4) was performed by dot blot affinity. In the first dot blot experiment, 2 µg of the specific Lys linked di-ubiquitin peptides (22, 23, 24), linear partial ubiquitin peptides containing a K63 or K48 residues (19, 20, 21), and the ubiquitin from bovine red blood cells as a control sample (18), were spotted on a nitrocellulose membrane. The membrane was incubated for 1 h with blocking buffer (Rotiblock). Then, the membrane was incubated with the primary ubiquitin antibody (K63-Ub antibody) in PBS-Tween (antibody:PBS-Tween ratio of 1:2500, v/v) for 1 hr. As detection antibody the horse radish peroxidase (HRP) goat anti-mouse in PBS-Tween (1:5000, v/v) was applied and incubated 1 hr. The membrane was developed by a mixture of ECL-solutions and exposed on a film in a dark room for 30 second (Fig. 79, Table 13). The results gave an intense positive response of specific Ub-K63 antibody to the ubiquitin peptides containing a Lys63 linkage site. The control bovine ubiquitin and the K48 linked di-ubiquitin used as negative control gave no response.
19 20 21
22 23 24
18
Ubiquitin Ub(61-76) Ub(54-76) Ub(48-76)
K63-Ub(61-76)2 K48-Ub(54-76)2 K63-Ub(48-76)2 30 Sec
Figure 79. Dot blots analysis of K63 linkage ubiquitin antibody to linear synthetic ubiqutin peptides (#II-IV) and lysine linked di-ubiquitin peptides (#V-VII).
Table 13. Different ubiquitin peptides contained K63 and K48 residues for dot-blot analysis.
Spot
Nr Peptide Sequence Dot
Blot*a
18 Ub(1-76) Ubiquitin from bovine (Sigma-Aldrich) -
19 Ub(61-76) H2N- 61IQ63KESTLHLVLRLRGG76 -COOH +
20 Ub (54-76) H2N-54RTLSDYNIQ63KESTLHLVLRLRGG76-COOH +
21 Ub(48-76) H2N-48KQLEDGRTLSDYNIQ63KESTLHLVLRLRGG76- OH -
22 K63-linked Ub(61-76)2
H2N- 61IQ63KESTLHLVLRLRGG76 –COOH
| H2N- 61IQKESTLHLVLRLRGG76
+
23 K63-linked Ub(54-76)2
H2N-54RTLSDYNIQ63KESTLHLVLRLRGG76-COOH ׀
H2N-54RTLSDYNIQ63KESTLHLVLRLRGG76
+
24 K48-linked Ub(48-76)2
H2N-48KQLEDGRTLSDYNIQ63KESTLHLVLRLRGG76-OH ׀
76GGRLRLVLHLTSEKQINYDSLTRGDELQK48-N2H
-
*a Dot blots analysis of Lys63 specific linkage ubiquitin antibody: (+), response and (-), no response
For the comparison of differences in affinity binding specificity of different ubiquitin Building Blocks to K63 linkage Ub-antibody an equimolar mixture, 100 µmol of each peptide, was used in an immuno-affinity method such as Dot blot and the affinity chromatography-MS analysis (MS data not shown). A Dot Blot experiment was carried out using the same affinity K63-Ub antibody with ubiquitin building blocks peptides.
The ubiquitin building blocks were prepared by SPPS and characterized by ESI-Ion trap MS (Table. 14). These ubiquitin peptides containing the specific lysine residues with an internal Gly-Gly dipeptide (“Building Blocks”; 25; Ub_K6-GG, 26; Ub_K11-GG, 27; Ub_K33-GG, 28; Ub_K48-GG and 29; Ub_K63-GG) were manually synthesized due to facilitate the correct coupling of each amino acid on the hydrophobic sequence. The choice of a side chain protecting group depends on the choice of protecting group on the α-amino group, so that differently acid sensitive or an “orthogonal” combination of protecting groups such as side chain protected lysine derivatives: Fmoc-Lys(Mtt)-OH or Dde-Lys(Fmoc)-OH are used. It is site-specific and allows only a single specific coupling reaction between the Cα-moiety of one peptide segment and the Nε-amine of another peptide segment (s. 3.2.3.2) [271].
The results of a correlation between structure and affinity interaction of the ubiquitin building blocks are presented in the Figure 80 and Table 14.
Ub_K6-GG Ub_K11-GG Ub_K33-GG
Ub_K48-GG Ub_K63-GG Ubiquitin 30 Sec
26 27
25
29 18
28
Figure 80. Dot blots analysis using monoclonal Ub-K63 antibody. (A), Dot blot experiment of specific lysine residues attached to a Gly-Gly dipeptide (25 - 29) and ubiquitin as a negative control (18).
Table 14. HPLC and Mass spectrometric characterization of ubiquitin sequences containing the specific lysine residues attached to Gly-Gly dipeptide which were tested by dot blot experiment with the Lys63 specific linkage ubiquitin antibody.
Nr Sample Sequence [M]calc/exp*a HPLC
Rt*b
Dot Blot*c
25 K6-GG NH2-MQIFVK-(GG)TLTG-CONH2 1251.7/
1250.53 30.5 +
26 K11-GG NH2-LTGK-(GG)TIT-CONH2 846.99/
846.60 28.0 -
27 K33-GG NH2-IQDK-(GG)EG-CONH2 801.85/
801.50 22.5 -
28 K48-GG NH2-LIFAGK-(GG)QLEDG-CONH2 1303.48/
1303.60 29.1 -
29 K63-GG NH2-SDYNIQK-(GG)ESTLHL-CONH2 1660.80/
1660.19 31.8 +
18* Mono-Ub Ubiquitin (1-76) 8564.85 - -
* Mono-ubiquitin from bovine red blood cells as a negative control was purchased from Sigma-Aldrich.
*a Average mass; ESI-Ion trap mass spectra were recorded on a Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a standard ESI source.
*b Analytical RP-HPLC columns: VydacTN C4 column (250 mm x 4.6 mm I.D.) with a 5 µm silica (300 Å pore size); eluents: 0.1 % TFA/water (A), 0.1% TFA/MeCN-water 80:20, v/v (B); flow rate: 1 mL/min; Gradient: 0 min 0 % B, 5 min 10 % B, 105 min 100 % B.
*c Dot blots analysis of Lys63 specific linkage ubiquitin antibody: (+), response and (-), no response.
The result of the test confirmed the specific binding of the K63-Ub antibody to the ubiquitin building blocks; Ub_K6-GG (25), and Ub_K63-GG (29). In contrast, for another ubiuitin Building Blocks containing the K11, K33 and K48 were observed no responses. By characterization of the secondary structure of ubiquitin sequences by circular dichroism spectroscopy, the specificity of K63-Ub antibody to the ubiquitin building blocks containing different Lys residues can be explained. The building block peptides; Ub_K6-GG and Ub_K63-GG in water showed the presence of a negative band around 202 nm (π-π*
transition) and a small negative shoulder around 230 nm, characteristic of an unordered structure. A beta sheet secondary structure was found for the K11and K48 linked partial ubqiutin peptides (building blocks; K11-GG, K48-GG) present a mixture secondary structure conformation between random coil and beta sheet (Fig. 81). The CD spectroscopy data indicated consistent results to reveal differences in binding specificities by the K63-Ub antibody, showing that antibody binding is influenced by antigen secondary structure.
200 220 240 260 -40
-30 -20 -10 0 10
Ellipticity
K6-GG K11-GG K48-GG K63-GG
Wavelength[nm]
Figure 81. Secondary structure determination of different partial ubiquitin sequences by circular dichroism spectroscopy. The ubiquitin peptides were dissolved in water at a concentration of 1 µ g/µl, and the measurements were carried at 25 ° C by averaging six scans between 190 and 260 nm.
2.5.5 Western Blot analysis of Lys63 linkage antibody with different Lys linked