2 research outputs found
ΠΡΠΎΠ±Π΅Π½Π½ΠΎΡΡΠΈ ΠΏΡΠΎΠ±ΠΎΠΏΠΎΠ΄Π³ΠΎΡΠΎΠ²ΠΊΠΈ Π»ΠΈΠ·Π°ΡΠΎΠ² Π΄Π»Ρ ΠΏΠΎΠ²ΡΡΠ΅Π½ΠΈΡ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ Π±Π΅Π»ΠΊΠΎΠ²ΡΡ ΠΏΠ°ΡΡΠ½Π΅ΡΠΎΠ² ΡΠ΅Π»Π΅Π²ΡΡ Π±Π΅Π»ΠΊΠΎΠ², ΠΊΠΎΠ΄ΠΈΡΡΠ΅ΠΌΡΡ Π³Π΅Π½Π°ΠΌΠΈ 18-ΠΎΠΉ Ρ ΡΠΎΠΌΠΎΡΠΎΠΌΡ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°
The aim of this work was to test modifications of the standard protocol for the sample preparation of cell/tissue lysate before performing the affinity isolation of lysate protein partners for the target protein (bait protein) which is covalently immobilized on an inert sorbent (e.g. BrCN-, SH-Sepharose 4B) or a carrier (e.g. paramagnetic nanoparticles). The series of our previous works on applying the approach to direct molecular fishing procedure with combination of affinity chromatography and LC-MS/MS analysis using a number of proteins, encoded by the genes of human chromosome 18, have shown that there are at least two problems affecting the specificity and the effectiveness of this procedure. These include: (i) redundancy of the background proteins in the eluates from an affinity sorbent (carrier) due to isolation of multiprotein complexes βlabeledβ with a direct protein partner which binds with a bait protein immobilized on the sorbent; (ii) low enrichment of the eluates with appropriate protein partners due to the fact that some direct protein partners in the lysate exist in stable βwild typeβ complexes with the bait protein itself. This means that latter group of protein partners will not be sufficiently isolated from lysate. Therefore, in order to increase the specificity and efficiency of affinity isolation of protein partners for the bait protein, we modified the standard protocol of lysate preparation and the preliminary step on dissociation of lysate protein complexes was added. Several model experiments for the choice of regeneration solution, assessment of their efficiency in the dissociation of lysate protein complexes as well as the stability and binding capacity of proteins were performed under the control of surface plasmon resonance (SPR) biosensor Biacore 3000 using HepG2 cell lysate. It was shown that acid treatment and incubation of the cell lysate for one min on ice (final lysate dilution 20 times) and subsequent neutralization (pH shift from 2.0 to 7.4) resulted in maximal dissociation of the lysate protein complexes without significant negative effects on the protein-protein interactions tested.Π¦Π΅Π»ΡΡ ΡΠ°Π±ΠΎΡΡ Π±ΡΠ»ΠΎ ΡΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠ°Π»ΡΠ½ΠΎΠ΅ ΡΠ΅ΡΡΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ ΠΌΠΎΠ΄ΠΈΡΠΈΠΊΠ°ΡΠΈΠΈ ΡΡΠ°Π½Π΄Π°ΡΡΠ½ΠΎΠ³ΠΎ ΠΏΡΠΎΡΠΎΠΊΠΎΠ»Π° ΠΏΡΠΎΠ±ΠΎΠΏΠΎΠ΄Π³ΠΎΡΠΎΠ²ΠΊΠΈ ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠ³ΠΎ/ ΡΠΊΠ°Π½Π΅Π²ΠΎΠ³ΠΎ Π»ΠΈΠ·Π°ΡΠ° ΠΏΠ΅ΡΠ΅Π΄ Π²ΡΠΏΠΎΠ»Π½Π΅Π½ΠΈΠ΅ΠΌ ΠΏΡΠΎΡΠ΅Π΄ΡΡΡ Π°ΡΡΠΈΠ½Π½ΠΎΠ³ΠΎ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ ΠΈΠ· Π½Π΅Π³ΠΎ Π±Π΅Π»ΠΊΠΎΠ²-ΠΏΠ°ΡΡΠ½Π΅ΡΠΎΠ² Π΄Π»Ρ ΡΠ΅Π»Π΅Π²ΠΎΠ³ΠΎ Π±Π΅Π»ΠΊΠ° (Π±Π΅Π»ΠΊΠ°-Π½Π°ΠΆΠΈΠ²ΠΊΠΈ), ΠΈΠΌΠΌΠΎΠ±ΠΈΠ»ΠΈΠ·ΠΎΠ²Π°Π½Π½ΠΎΠ³ΠΎ Π½Π° ΠΈΠ½Π΅ΡΡΠ½ΠΎΠΌ ΡΠΎΡΠ±Π΅Π½ΡΠ΅ ΠΈΠ»ΠΈ ΠΏΠ°ΡΠ°ΠΌΠ°Π³Π½ΠΈΡΠ½ΡΡ
Π½Π°Π½ΠΎΡΠ°ΡΡΠΈΡΠ°Ρ
. Π¦ΠΈΠΊΠ» Π½Π°ΡΠΈΡ
ΠΏΡΠ΅Π΄ΡΠ΄ΡΡΠΈΡ
ΡΠ°Π±ΠΎΡ, ΠΏΠΎΡΠ²ΡΡΠ΅Π½Π½ΡΡ
ΠΏΡΡΠΌΠΎΠΌΡ ΠΌΠΎΠ»Π΅ΠΊΡΠ»ΡΡΠ½ΠΎΠΌΡ ΡΠΈΡΠΈΠ½Π³Ρ Ρ ΡΠΎΠΏΡΡΠΆΠ΅Π½ΠΈΠ΅ΠΌ Ρ
ΡΠΎΠΌΠ°ΡΠΎΠ³ΡΠ°ΡΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΈ ΠΌΠ°ΡΡ-ΡΠΏΠ΅ΠΊΡΡΠΎΠΌΠ΅ΡΡΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΌΠ΅ΡΠΎΠ΄ΠΎΠ² ΠΈ ΡΠ΅Ρ
Π½ΠΎΠ»ΠΎΠ³ΠΈΠΈ ΠΏΠ°ΡΠ°ΠΌΠ°Π³Π½ΠΈΡΠ½ΡΡ
Π½Π°Π½ΠΎΡΠ°ΡΡΠΈΡ c ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ ΡΡΠ΄Π° Π±Π΅Π»ΠΊΠΎΠ² 18-ΠΎΠΉ Ρ
ΡΠΎΠΌΠΎΡΠΎΠΌΡ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° ΠΈ ΡΠ°ΠΊΠΆΠ΅ Π΄ΡΡΠ³ΠΈΡ
Π±Π΅Π»ΠΊΠΎΠ² ΠΏΠΎΠΊΠ°Π·Π°Π», ΡΡΠΎ ΡΡΡΠ΅ΡΡΠ²ΡΡΡ, ΠΏΠΎ ΠΊΡΠ°ΠΉΠ½Π΅ ΠΌΠ΅ΡΠ΅, Π΄Π²Π΅ ΠΏΡΠΎΠ±Π»Π΅ΠΌΡ, Π²Π»ΠΈΡΡΡΠΈΠ΅ Π½Π° ΡΠΏΠ΅ΡΠΈΡΠΈΡΠ½ΠΎΡΡΡ ΠΈ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΡ Π΄Π°Π½Π½ΠΎΠΉ ΠΏΡΠΎΡΠ΅Π΄ΡΡΡ: (i) ΠΈΠ·Π±ΡΡΠΎΡΠ½ΠΎΡΡΡ ΡΠΎΠ½ΠΎΠ²ΡΡ
Π±Π΅Π»ΠΊΠΎΠ² Π² ΡΠ»ΡΠ°ΡΠ°Ρ
Ρ Π°ΡΡΠΈΠ½Π½ΠΎΠ³ΠΎ ΡΠΎΡΠ±Π΅Π½ΡΠ°, ΠΎΠ±ΡΡΠ»ΠΎΠ²Π»Π΅Π½Π½Π°Ρ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΠ΅ΠΌ ΠΌΡΠ»ΡΡΠΈΠ±Π΅Π»ΠΊΠΎΠ²ΡΡ
ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠΎΠ², ΠΌΠ΅ΡΠ΅Π½ΡΡ
ΠΏΡΡΠΌΡΠΌ ΠΏΠ°ΡΡΠ½Π΅ΡΠΎΠΌ, ΠΊΠΎΡΠΎΡΡΠΉ ΡΠ²ΡΠ·ΡΠ²Π°Π΅ΡΡΡ Ρ ΡΠ΅Π»Π΅Π²ΡΠΌ Π±Π΅Π»ΠΊΠΎΠΌ Π½Π° ΡΠΎΡΠ±Π΅Π½ΡΠ΅; (ii) Π½ΠΈΠ·ΠΊΠ°Ρ ΠΎΠ±ΠΎΠ³Π°ΡΠ΅Π½Π½ΠΎΡΡΡ ΡΠ»ΡΠ°ΡΠΎΠ² Π±Π΅Π»ΠΊΠ°ΠΌΠΈ-ΠΏΠ°ΡΡΠ½Π΅ΡΠ°ΠΌΠΈ ΡΠ΅Π»Π΅Π²ΠΎΠΉ Π³ΡΡΠΏΠΏΡ ΠΎΠ±ΡΡΠ»ΠΎΠ²Π»Π΅Π½Π½Π°Ρ ΡΠ΅ΠΌ, ΡΡΠΎ ΡΠ° ΠΈΠ»ΠΈ ΠΈΠ½Π°Ρ ΡΠ°ΡΡΡ ΠΏΡΡΠΌΡΡ
Π±Π΅Π»ΠΊΠΎΠ²-ΠΏΠ°ΡΡΠ½Π΅ΡΠΎΠ² Π² Π»ΠΈΠ·Π°ΡΠ΅ Π½Π°Ρ
ΠΎΠ΄ΠΈΡΡΡ Π² ΡΠΎΡΡΠ°Π²Π΅ ΡΡΠ°Π±ΠΈΠ»ΡΠ½ΡΡ
ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠΎΠ² Β«Π΄ΠΈΠΊΠΎΠ³ΠΎ ΡΠΈΠΏΠ°Β» Ρ ΡΠ°ΠΌΠΈΠΌ Π±Π΅Π»ΠΊΠΎΠΌ-Π½Π°ΠΆΠΈΠ²ΠΊΠΎΠΉ ΠΈ Π½Π΅ Π±ΡΠ΄Π΅Ρ Π² Π΄ΠΎΡΡΠ°ΡΠΎΡΠ½ΠΎΠΉ ΡΡΠ΅ΠΏΠ΅Π½ΠΈ Π²ΡΠ΄Π΅Π»Π΅Π½Π° ΠΈΠ· Π»ΠΈΠ·Π°ΡΠ°. ΠΠΎΡΡΠΎΠΌΡ Π΄Π»Ρ ΠΏΠΎΠ²ΡΡΠ΅Π½ΠΈΡ ΡΠΏΠ΅ΡΠΈΡΠΈΡΠ½ΠΎΡΡΠΈ ΠΈ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π°ΡΡΠΈΠ½Π½ΠΎΠ³ΠΎ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ Π±Π΅Π»ΠΊΠΎΠ²-ΠΏΠ°ΡΡΠ½Π΅ΡΠΎΠ² ΡΠ΅Π»Π΅Π²ΠΎΠ³ΠΎ Π±Π΅Π»ΠΊΠ° Π½Π°ΠΌΠΈ ΠΏΡΠ΅Π΄Π»ΠΎΠΆΠ΅Π½Π° ΠΌΠΎΠ΄ΠΈΡΠΈΠΊΠ°ΡΠΈΡ ΡΡΠ°Π½Π΄Π°ΡΡΠ½ΠΎΠΉ ΠΏΡΠΎΠ±ΠΎΠΏΠΎΠ΄Π³ΠΎΡΠΎΠ²ΠΊΠΈ, Π·Π°ΠΊΠ»ΡΡΠ°ΡΡΠ°ΡΡΡ Π² ΠΏΡΠ΅Π΄Π²Π°ΡΠΈΡΠ΅Π»ΡΠ½ΠΎΠΉ Π΄ΠΈΡΡΠΎΡΠΈΠ°ΡΠΈΠΈ Π±Π΅Π»ΠΊΠΎΠ²ΡΡ
ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠΎΠ² Π»ΠΈΠ·Π°ΡΠ°. ΠΠΎΠ΄Π΅Π»ΡΠ½ΡΠ΅ ΡΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΡ ΠΏΠΎ Π²ΡΠ±ΠΎΡΡ ΡΠ΅Π³Π΅Π½Π΅ΡΠ°ΡΠΈΠΎΠ½Π½ΠΎΠ³ΠΎ ΡΠ°ΡΡΠ²ΠΎΡΠ°, ΠΎΡΠ΅Π½ΠΊΠ΅ ΡΡΠ°Π±ΠΈΠ»ΡΠ½ΠΎΡΡΠΈ ΠΈ ΡΠ²ΡΠ·ΡΠ²Π°ΡΡΠ΅ΠΉ ΡΠΏΠΎΡΠΎΠ±Π½ΠΎΡΡΠΈ Π±Π΅Π»ΠΊΠΎΠ² ΠΏΡΠΈ Π΅Π³ΠΎ Π²ΠΎΠ·Π΄Π΅ΠΉΡΡΠ²ΠΈΠΈ, Π° ΡΠ°ΠΊΠΆΠ΅ ΠΎΡΠ΅Π½ΠΊΠ° ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π΄ΠΈΡΡΠΎΡΠΈΠ°ΡΠΈΠΈ ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠΎΠ² Π² Π»ΠΈΠ·Π°ΡΠ΅ Π±ΡΠ»ΠΈ Π²ΡΠΏΠΎΠ»Π½Π΅Π½Ρ ΠΏΠΎΠ΄ ΠΊΠΎΠ½ΡΡΠΎΠ»Π΅ΠΌ ΠΎΠΏΡΠΈΡΠ΅ΡΠΊΠΎΠ³ΠΎ Π±ΠΈΠΎΡΠ΅Π½ΡΠΎΡΠ° Biacore 3000 (Β«GE HealthcareΒ», Π‘Π¨Π) Ρ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ Π»ΠΈΠ·Π°ΡΠ° ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠΉ ΠΊΡΠ»ΡΡΡΡΡ Π³Π΅ΠΏΠ°ΡΠΎΠΊΠ°ΡΡΠΈΠ½ΠΎΠΌΡ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° (HepG2) ΠΈ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΡ
ΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠΎΠ² Π±Π΅Π»ΠΊΠΎΠ², ΠΊΠΎΠ΄ΠΈΡΡΠ΅ΠΌΡΡ
Π³Π΅Π½Π°ΠΌΠΈ 18-ΠΎΠΉ Ρ
ΡΠΎΠΌΠΎΡΠΎΠΌΡ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°, ΠΠΎΠΊΠ°Π·Π°Π½ΠΎ, ΡΡΠΎ ΠΊΠΈΡΠ»ΠΎΡΠ½Π°Ρ ΠΎΠ±ΡΠ°Π±ΠΎΡΠΊΠ° ΡΠ°Π·Π±Π°Π²Π»Π΅Π½Π½ΠΎΠ³ΠΎ Π² 20 ΡΠ°Π· Π»ΠΈΠ·Π°ΡΠ° Ρ ΠΊΡΠ°ΡΠΊΠΎΠ²ΡΠ΅ΠΌΠ΅Π½Π½ΠΎΠΉ ΡΠΊΡΠΏΠΎΠ·ΠΈΡΠΈΠ΅ΠΉ Π² ΡΠ΅ΡΠ΅Π½ΠΈΠ΅ 1 ΠΌΠΈΠ½ Π½Π° Π»ΡΠ΄Ρ ΠΈ Ρ ΠΏΠΎΡΠ»Π΅Π΄ΡΡΡΠ΅ΠΉ Π½Π΅ΠΉΡΡΠ°Π»ΠΈΠ·Π°ΡΠΈΠ΅ΠΉ (Ρ ΡΠ 2.0 Π΄ΠΎ ΡΠ 7.4) ΠΏΡΠΈΠ²ΠΎΠ΄ΠΈΠ»Π° ΠΊ ΠΌΠ°ΠΊΡΠΈΠΌΠ°Π»ΡΠ½ΠΎΠΉ Π΄ΠΈΡΡΠΎΡΠΈΠ°ΡΠΈΠΈ Π±Π΅Π»ΠΊΠΎΠ²ΡΡ
ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠΎΠ² Π»ΠΈΠ·Π°ΡΠ°, Π½Π΅ ΠΎΠΊΠ°Π·ΡΠ²Π°Ρ ΡΡΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΎΠ³ΠΎ Π½Π΅Π³Π°ΡΠΈΠ²Π½ΠΎΠ³ΠΎ Π²Π»ΠΈΡΠ½ΠΈΡ Π½Π° ΡΠ΅ΡΡΠΈΡΡΠ΅ΠΌΡΠ΅ Π±Π΅Π»ΠΎΠΊ- Π±Π΅Π»ΠΊΠΎΠ²ΡΠ΅ Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΡ