The overall goal of this procedure is to biochemically reconstitute a steroid receptor HSB 90 protein complex and reactivate steroid receptor ligand binding activity, which can be used to test HSB 90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. This is accomplished by first preparing cell cytosol containing functional glucocorticoid receptors. Next, the GR from the cell cytosol is immuno absorbed and endogenous.
HSP 90 is dissociated. Then the GR HSP 90 hetero complex is reconstituted using an exogenous source of HSP 90 and other chaperone proteins and co-factors. Finally, the reconstituted protein complexes and ligand binding activity are analyzed specifically methods such as SDS page, western blotting and radioactive ligand binding assays are used to show GR HSP 90 hetero complex formation and steroid binding activity.
This method can help answer key questions in the HSP 90 molecular chaperone field, such as what cofactor chaperones and COS chaperones are required to facilitate HSP 90 functional activity. Though this method can provide insight into protein folding, it can also be applied to other systems such as developing HSP 90 dependent lead compounds and analysis of HSP 90 needed intracellular signaling pathways Demonstrating the procedure will be Hannah Franklin and Nathan for AWA two recently graduated students from my laboratory To isolate functional glucocorticoid receptor or GR grow mouse fibroblast L 9 29 cells or SF nine cells that have been infected with recombinant GR bao virus and are in the exponential growth phase. Centrifuge the cell suspension at 5, 000 Gs for five minutes at four degrees Celsius.
The resulting pellet should be about one to five milliliters in volume. Wash the cell pellet three times with 15 milliliters of hank's buffered saline solution. Prepare 1.5 volumes of homogenization buffer containing HEM buffer one millimolar phenyl methyl syl fluoride or PMSF and three complete mini protease inhibitor tablets ground using 50 strokes of a downs.
Homogenizer ruptured the cells, performed the step on ice to prevent protein degradation. Ultracentrifuge the homogenate at 100, 000 Gs for 30 minutes at four degrees Celsius. Aliquot the super natin containing the cytosol into 1.5 milliliter micro centrifuge tubes of 500 microliters each and store at minus 20 degrees Celsius.
To prepare a mixture containing a monoclonal IgG antibody raised against the receptor specific GR combine 100 microliters of thaw GR containing cytosol. 200 microliters of TEGM buffer, 100 microliters of a 20%protein asero or PAS slurry and five micrograms of an anti G monoclonal IgG antibody such as bugger two. Due to the relatively large PAS bead size.
Use a cut P 200 pipette tip to transfer PAS from the 20%slurry to sample tubes. Place the samples on a vertical rotating wheel and incubate for a minimum of two hours with constant rotation. Next, remove the unbound proteins from the antibody PAS pellet by spinning the samples in a refrigerated centrifuge for one minute.
At 10, 000 Gs, wash the pellet twice by adding one milliliter of TEGM buffer vortexing, then spinning. Remove the final wash using a crimped P 200 pipette tip to dissociate endogenous HSB 90 from the GR immuno pellet and 355 microliters of TEG buffer and 45 microliters of five molar sodium chloride to the antibody. PAS pellet previously prepared.
Place samples on a vertical rotating wheel and to incubate for one and a half hours with constant rotation. Remove the unbound proteins from the immuno pellet by spinning them for one minute at 10, 000 Gs.Wash the pellets once with one milliliter of TEG buffer and once with one milliliter of 10 millimolar heaps. Buffer pH 7.4 Using a crimped P 200 pipette tip, remove the wash, taking care not to disturb the pellet.
To reconstitute the GR HSB 90 hetero complex, add to the pellet a mixture containing 50 microliters of a molecular chaperone source, such as a rabbit reticulocyte, lysate, and five microliters of an A TP generating system System. This next step is critical to ensure adequate mixing of the pellet and reconstitution solution. Incubate the samples in a 30 degree Celsius water bath for exactly 20 minutes.
Flick the tubes every one to two minutes in order to gently disturb the pellet and mix the reconstitution solution. Add one milliliter of TEGM buffer vortex and centrifuge at 10, 000 Gs.For one minute. Remove the supernatant being careful not to disturb the pellet.
Repeat the wash two more times to identify proteins in the reconstituted pellet. Prepare samples for electrophoresis by adding 50 microliters of SDS page sample buffer containing betta mercaptoethanol v. Text the samples then incubate for five minutes in a boiling water bath or a 100 degree Celsius electronic heat block.
Centrifuge the samples at 10, 000 Gs for one minute. Use the supernatant from these samples for SDS page or microfluidic electrophoresis followed by western blot. Using a P 200 crimped tip to load the supinate will limit accidental aspiration of the pellet.
The next step is to assay the GR HSP 90 protein complex for steroid binding activity. Using a tritiated steroid begin by adding to the reconstituted immuno pellet. 47.5 microliters of HEM buffer and 2.5 microliters of two micromolar tritiated dexamethasone.
Gently mix the pellet being careful to minimize the amount of sample displaced to the wall of the micro centrifuge tube. Incubate the sample overnight undisturbed on ice. Add one milliliter of TEGM buffer gently mix and centrifuge at 10, 000 GS for one minute.
Discard the supernat being mindful. It contains unbound tritiated steroid repeat for a total of three washes with TEGM buffer completely. Remove all supernatant at the conclusion of the final wash.
Using a crimped P 200 pipette tip. Suspend the washed immuno pellets in 200 microliters of TEGM buffer and transfer to 10 milliliters. Scintillation vials.
Add 4.8 milliliters of scintillation cocktail to the vials and vortex. Finally, carry out liquid scintillation spectrometry representative SDS page and western blot data are shown here in this kumasi stain gel. The endogenous GR HSB 90 hetero complex is immuno absorbed from the cell cytosol using anti GR antibodies and individual proteins that coun absorbed with the GR are visualized shown.
Here are Experian microfluidic electrophoresis data representing reconstituted GR HSP 90 hetero complexes. The specificity of the GR immuno absorption is confirmed by replacing the immuno ads absorbing anti GR antibody with a non-immune antibody even when excessive amounts are used. No GR or molecular chaperones are immuno precipitated.
Likewise, it is possible to confirm the dissociation of HSP 90 and other chaperone proteins from the salt stripped GR by western blo of the stripped and reconstituted immuno pellets. The heavy chain of the immuno absorbing antibody is detectable by both kumasi stain and western blotting, and it confirms that equal sized immuno pellets are being visualized in each lane. Representative steroid binding activity data of the immuno absorptive GR are presented here using either reticulocyte lysate or purified proteins.
Steroid binding activity of the reconstituted GR HSB 90 hetero complex typically returns to 75 to 100%of the endogenous GR HSB 90 binding activity level. Similar to the electrophoresis data, a non-immune antibody may be used in place of the anti GR antibody as a negative control and as a measure of non-specific tritiated steroid binding. Following this procedure, other methods like 2D gel electrophoresis can be performed in order to answer additional questions like what impact HSP 90 post-translational modifications has on hetero complex reconstitution and steroid binding Reconstitution assays, such as the ones described here, have allowed researchers to explore the functional significance of the HSP 90 HSP 70 based MultiPro chaperone machinery and its stepwise assembly using the glucocorticoid receptor as a model HSP 90 client protein.
After watching this video, you should have a good understanding of how to prepare functional glucocorticoid receptor HSP 90 protein complexes from purified proteins in cellular lysates. The steps used include preparing GR containing cell lysate receptor immune absorption, salt stripping of the endogenous proteins and reconstitution of the hetero complex using exogenous HS P 90, HSP 70 cofactors and cos chaperones.
