What is the reason for the false positive result of GST-Pull down experiment and how to solve it?
A: Two DNA-binding proteins that do not interact with each other may show false positive results of interaction due to the presence of DNA. Therefore, the step of adding nuclease is necessary when performing the GST pull-down assay
What are the factors that affect the results of GST-Pull down experiments and how to solve them?
A: (1) The first step of the experiment is the fusion expression of the GST-tagged fusion protein, then the selection of the expression vector is an important factor. When selecting the vector, we have to consider factors such as the amount of protein expressed by the vector and whether the expression can be induced by the inducer. Our commonly used vector is pGEX-4T-1.
(2) Soluble expression temperature is also a crucial factor when performing induced expression of proteins. When the temperature is too high, the vector tends to form inclusion bodies. If the complexation is not smooth and the protein is inactive, it will affect the subsequent experiments. An induction temperature mapping is recommended, with a common induction temperature of 16℃~25℃.
(3) The time of induction is also very important. The best time to add inducer to induce expression is when the bacteria are in logarithmic growth phase. If the induction time is too long, the inducer will not be metabolized by the bacteria and will keep carrying out the process of inducing protein expression. The concentration of the inducer, the time of induction, and the temperature of incubation are all factors that combine to influence the results of protein expression.
Why are the bands of gel run out inconsistent for the same batch of extracted protein?
A: Several reasons may be involved: (1) The gel moved wrongly when pulling the comb and seeped into other wells or leaked out after adding the sample. Because the internal reference expression is high, it may not show the difference.
(2) Unevenness when adding samples may produce errors.
(3) Preparation of gel should ensure uniformity without air bubbles and clean glass plates so as to ensure consistent and reliable results.
What if the proteins in the sample are degraded by protease?
A: If the proteins in the sample are degraded by protease, it is recommended to add protease inhibitor during the operation. If possible, the entire operation should be performed at 4°C to alleviate the protein degradation in the sample.
Why is there a stray band on the CBB plot of the experimental results, does it affect the results?
A: No, it does not affect the results. Generally, the purity of the purified protein small sample provided is >70%. Some protein purification process will have some possible intercalated proteins, degraded proteins, which cannot be removed by affinity chromatography. This does not affect the subsequent pull-down results analysis.
Do the target proteins need to be codon optimized?
A: There is a preference for the use of codons in different species, and codon optimization facilitates the expression of eukaryotic proteins in E. coli.
What should I do if the binding efficiency of the target protein is low or does not bind?
A: 1) Protein sequence with shift code and other errors
Sequencing to confirm, adjust the reading frame to obtain GST.Tag fusion expression protein. Select a suitable host bacterium, such as protease-deficient E. coli, rare codon host bacterium Rosetta series.
2) The fusion protein may have changed the conformation of the GST, therefore reducing the binding ability of the GST-tagged protein.
Detection of GST binding in the pGEX vector used. Prepare a cell sonication lysate with the pGEX used and assay its binding to the chromatography column. If the binding is good, it is possible that the tagging protein has changed the conformation of the GST and reduced the affinity of the GST tagging protein. The results can be improved by lowering the binding temperature to 4°C to limit the washing.
3) Aggregation precipitation of GST fusion proteins occurs
Add DTT to the lysis buffer and other buffer systems. 1-20 mM DTT significantly increases the binding of some GST fusion proteins.
4) GST fusion proteins are over-diluted
Re-concentrate the sample to increase the protein concentration.
5) GST fusion proteins are denatured by mechanical lysis methods (e.g. sonication)
Excessive ultrasound heat production can destroy the tag protein, and ultra-high pressure fragmentation is recommended.
6) Unsuitable binding buffer conditions
Below pH 6.5 or above pH 8, the fusion protein does not bind sufficiently to the chromatography column resulting in low binding efficiency. Confirm that the magnetic beads are fully equilibrated by pH6.5~8.0 buffer before being used for purification of the target protein.
7) The chromatographic column is used too many times and impurities interfere
Regenerate the chromatographic column or use new chromatographic column.
What causes the target protein not to elute down or the elution rate to be low?
A: 1) The volume of elution buffer is too small
Increase the volume of elution buffer.
2) The pH of the elution buffer is too low
Adjust the pH of the elution buffer to 8-9.
3) The elution time is not enough
Increase the time of elution buffer reacting with magnetic beads.
4) The concentration of glutathione in the elution buffer is too low
Increase the concentration of glutathione in the elution buffer.
5) Glutathione in elution buffer fails to be oxidized
The elution buffer is prepared and used now.
(6) The ionic strength of the elution buffer is too low
Increase the ionic strength in the elution buffer, adding 0.1~0.2M sodium chloride in the elution buffer will also improve the elution effect.
Why do the eluted proteins contain impurities?
A: 1) Protein is degraded in the host bacteria
Use protease-deficient strains lon- or ompT, or double-deficient strains of ompT and lon.
2) GST fusion protein is partially degraded by protease
Add protease inhibitor PMSF.
3) Excessive sonication during cell fragmentation
Decrease the lysis time and the addition of lysozyme before mechanical lysis may lead to better results. Avoid foaming, as this may denature the labeled protein. Over-cleavage can also lead to co-purification of host cell proteins and GST-tagged proteins.
4) Molecular chaperones may be co-purified
Excess bands may be caused by co-purification of some molecular chaperones. These molecular chaperones are involved in the correct folding of the newly generated proteins in E. coli. For example: DnaK (molecular weight 70 000), DnaJ (molecular weight 37 000), GrpE (molecular weight 40 000) GroEL (molecular weight 57 000), GroES (molecular weight 10 000). Several methods to isolate GST fusion proteins from these co-purified proteins have been published.
How to solve the problem of multiple bands found in electrophoretic detection after enzymatic cleavage of the target protein?
A: Proteolytic cleavage occurs within the host bacterium.
Probe to detect when the bands appear. If it is determined that the excess bands were not present prior to cleavage by Precission Protease, thrombin, and coagulation factor Xa, these bands may be the result of degradation in the host bacterium. The target protein may contain cleavage sites for Precission Protease, Thrombin, Coagulation Factor Xa, please check the sequence.
Difference between Co-IP and Gst pull-down?
A: Co-IP utilizes the specificity of the antigen-antibody reaction. GST pull down generally uses a recombinant protein with a GST tag, incubates it with the target protein, and finally pulls down the interacting complex with GST-bound beads.
