BCA assay
Firstly, a BCA assay was performed on all the expressed proteins, and samples concentrations were determined by comparing their assay response to that of a series of dilutions of a standard protein with known concentrations, the used standard protein in the assay was BSA with dilutions; 2mg/ml, 1mg/ml, 0.5mg/ml. 0.25mg/ml, and 0.125mg/ml.
Fig.1: The standard curve to which our proteins concentrations were determined against
GST-DoCs
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Fig.2: BCA assayed GST-DoCS-induced samples compared to non-induced ones.
Results:
- The estimated concentration = 0.406 mg/mL which shows a successful induction and extraction.
His-CoH2
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Fig.3: BCA assayed His-CoH2 induced samples compared to non-induced ones.
Results:
- The estimated concentration = 0.287 mg/ml which shows a successful induction and extraction.
Next His-CoH2 was purified with affinity chromatography’s, then quantified by BCA.
Fig.4:BCA for the affinity purified His-CoH2. E1; elution 1, E2; elution 2, W1; wash 1, W2; wash 2
Comment:
From the shown graph: E1 and E2 have the same concentration. So, we troubleshoot this case that we may have a problem with our elution buffer, elution volume, or imidazole concentration.
His-Tau
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Fig.5: BCA assayed His-Tau induced sample compared to non-induced ones.
Results:
- The estimated concentration = 0.2867 mg/ml which shows a successful induction and extraction.
Next His-Tau was purified with affinity chromatography’s, then quantified by BCA.
Fig.6: BCA for the affinity purified His-Tau. E1; elution 1, E2; elution 2, W1; wash 1, W2; wash 2
Comment:
From the shown graph: E2 concentration Is much lower than E1 indicating a successful purification.
His-trim-L-Doc
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Fig.7: BCA assayed His-TRIM21-L-DoCS induced sample compared to non-induced ones.
Results:
The estimated concentration = 0.211 mg/ml which shows a successful induction and extraction.
Next His-TRIM21-L-DoCS was purified with affinity chromatography’s, then quantified by BCA.
Fig.8: BCA for the affinity purified His-TRIM21-L-DoCS. E1; elution 1, E2; elution 2, W1; wash 1, W2; wash 2
Comment:
From the shown graph: E2 concentration Is slightly lower than E1 indicating a successful purification.
GST-Coh2
This part was extracted using the “chemical lysis” protocol in the experiments Page
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Fig.9: BCA assayed GST-CoH2 induced sample compared to non-induced ones.
Results:
- The estimated concentration = (0.1175 ± 0.02050) mg/ml which shows a successful induction and extraction.
His-Trim
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Fig.10: BCA for the affinity purified His-TRIM21. E1; elution 1, E2; elution 2, W1; wash 1, W2; wash 2
Comment:
From the shown graph: E2 concentration is slightly lower than E1 indicating a successful purification.
His- Doc
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Fig.11: BCA assayed His-DoCS induced sample compared to non-induced ones, the assay shows a remarkable difference between the induced and non-induced samples
Results:
- The estimated concentration = 0.8574 mg/ml which shows a successful induction and extraction.
Next His-DoCS was purified with affinity chromatography’s, then quantified by BCA.
Fig.12: BCA for the affinity purified His-DoCS. E1; elution 1, E2; elution 2, W1; wash 1, W2; wash 2
Comment:
From the shown graph: E2 concentration Is lower than E1 indicating a successful purification.
GST-Coh-Linker-WWW
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Fig.13: BCA assayed GST-CoH2-L-WWW induced sample compared to non-induced ones.
Results:
The estimated concentration = 0.7195 mg/ml which shows a successful induction and extraction.
GST-Coh-Linker-TD28REV
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Fig.14: BCA assayed GST-CoH2-L-TD28REV induced sample compared to non-induced ones.
Results:
The estimated concentration = 0.4976 mg/ml which shows a successful induction and extraction.
UBE2N
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Then performing affinity chromatography and undergoing BCA assay.
Fig.15: BCA for the affinity purified His-UBE2N. E1; elution 1, E2; elution 2, W1; wash 1, W2; wash 2
Comment:
From the shown graph: E2 concentration Is lower than E1 indicating a successful purification.
UBE2W
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Then performing affinity chromatography and undergoing BCA assay.
Fig.16: BCA for the affinity purified His-UBE2W. E1; elution 1, E2; elution 2, W1; wash 1, W2; wash 2
Comment:
From the shown graph: E2 concentration Is lower than E1 indicating a successful purification.
UBE2V2
This part was extracted using the “chemical lysis” protocol in the experiments Page.
The induced samples were compared with the non-induced samples to estimate the protein concentration.
Then performing affinity chromatography and undergoing BCA assay.
Fig.17: BCA for the affinity purified His-UBE2N. E1; elution 1, E2; elution 2, W1; wash 1, W2; wash 2
Comment:
From the shown graph: E2 concentration Is lower than E1 indicating a successful purification.
Pull-down assay
Pulldown assay
The pull-down technique is a method for performing ligand binding assays with recombinant proteins capable of binding to an affinity matrix in the presence or absence of a denaturing agent (that can be replaced by a physiological buffer). In order to examine direct protein-protein interactions, the method involves coupling a known quantity of protein to a given quantity of affinity matrix and then employing the suspension of this protein-coupled matrix as the source of the recombinant protein. This will entail using a pure protein with a tag as "bait" to bind any associated proteins (prey). The first step of the process is to immobilize the tagged protein (DOCs) on an affinity ligand specific to the tag. Next, affinity support is built to capture and purify other proteins (COH2) that interact with the immobilized protein (DOCs). Interaction complexes are eluted using an affinity ligand-specific eluting solution after the prey (COH2) proteins and an immobilized bait (DOCS) protein have been incubated. The binding will be determined using SDS-PAGE. (Sinha et al.,1994) (Louche et al.,2017)
1] DOCs &CoH2 interaction
DOCs and CoH2 were expressed two times fused with two different tags (His-tag and Gst-tag) in Bl21 (DE3); where pulldown assay was preformed two times to test the binding affinity between our PROTAC domains as follows:
1. Purified His-CoH2 against the total cell lysate containing GST-DOCs.
2. Purified His-DOCs against the total cell lysate containing His-CoH2.
Then, the binding affinities were compared and analyzed using BCA assay.
_Different tags were used to test the effect of tags on expression yield and binding affinities of the domains.
Fig.18:Using BCA assay to measure the concentration proteins after interaction Between His-coh2 & Gst-DOCs and HIS-DOCs &Gst-coh2
Data analysis of the effect of the interaction between His-CoH2 and GST-DoCS
Data analysis of the effect of the interaction between His-CoH2 and GST-DoCS
Comment:
The interaction has successfully happened using GST-DoCS against His-CoH2, while no binding was seen using His-DoCS due to its instability and low protein yielded.
2] [GST-COH-TBP] against [His-Tau]
Tau was expressed tagged with His-tag, aggregated and then its affinity was tested against the system targeted binding peptides namely, TDrev28 and WWW, the peptides were GST-tagged and fused with CoH2 domain. This allowed us to establish the binding affinity using a pulldown test.
Fig.19: Using BCA assay to measure the concentration of proteins after interaction and compare the affinity of the aggregated tau against the two different peptides (TD28 rev and WWW).
Data analysis of the effect of the interaction between aggregated tau and GST-CoH2-TD28rev
Data analysis of the effect of the interaction between aggregated tau and GST-CoH2-WWW
Comment:
The interaction has successfully happened using both peptides against His-tau, with higher binding affinities between tau and GST-CoH2-TD28rev.
Fig.20: Gel after pull-down assay shows interaction between (Tau aggregates) and the binding peptides (TD28 rev) have been successfully happened
3] [TRIM-L-DOC] against [COH-TBP]
TRIM21 was expressed fussed with DOC to confirm the binding affinity between [TRIM-DOC] and expressing the [COH-TBP]. The TBP by its two types (TDrev28 or WWW). We can check the binding affinity using a pulldown experiment to confirm the interaction between his-Trim-linker-DOC and GST-COH-TBP and analyze them using BCA.
Fig.21: Using BCA assay to measure the concentration proteins after interaction Between (Trim-L-DOCs) and two binding peptides (TD28 rev and WWW).
Data analysis of the effect of the interaction between His-TRIM21-DoCS and GST-CoH2-TD28rev
Data analysis of the effect of the interaction between tau aggregated against His-TRIM21-DoCS linked with GST-CoH2-WWW
Comment:
The interaction has successfully happened with higher results for binding between tau aggregates and www binding peptide.
[4] Trim-21 system (His-Trim-L-DOCs &Gst-coh2-WWW) vs Tau aggregates.
the result of pull-down assay show that the whole system interacts successfully with the tau aggregates inducing the ubiquitin to recruit the 26s proteosome for degrading these aggregates.
Fig.22: Using BCA assay to measure the concentration proteins after interaction Between (Our whole system) and (tau aggregates) have been successfully happened so our system is efficient to bind to aggregates.
Comment:
The result of pull-down assay show that the whole system interacts successfully with the tau aggregates.
SDS-PAGE
Well number 1: non-induced GST-coh2-www
Well number 2: non-induced GST-coh2-TD28rev
Well number 3: induced GST-coh2-www
Well number 4: induced GST-coh2-TD28rev
Comment
The results of SDS-PAGE show that in case of induction (well 3&4 respectively) GST-coh2-www and GST-coh2-TD28rev appear as clear bands so, induction done successfully while in case of non-induction well 1&2 the bands are faint.
Well 1: induced tau monomer appeared at 50 and 75 kDa
Well 2: non-induced tau monomer
Well 3: tau aggregates found between 100 to 200 kDa after aggregation with heparin
Comment
The results of SDS-PAGE showed that induction of tau monomers done successfully as it found in well 1 between 50 and 75 kDa and in case of forming aggregation of tau by heparin the band shown that heparin made different sizes of aggregation as the tau aggregates appears ranged from 100 to 200.
Well 1: induced Trim-linker-Docs
Well 2: non-induced Trim-linker-Docs
Comment
The results of SDS-PAGE show that induction of trim linked to dockerin done successfully as shown in well 1 while the non-induced one appears faint.
Well 1: induced His-Docs
Well 2: 0hr induced His-Docs
Well 3: non-induced His-Docs
Comment
The results of SDS-PAGE show that induction of his linked to dockerin done successfully as shown in well 1 and 0hr induction not appear clear as induced one while the non-induced appears faint.