Basic Protocol: Testing and use of TimeSTAMP tags by immunoblotting
Immunoblotting provides a straightforward way to test that the TimeSTAMP cassette is operating properly when fused to a protein of interest. It also provides a quantitative method for measuring rates of protein synthesis from a reporter construct within a defined time window under different stimulation or treatment conditions.
BILN-2061 (Boehringer Ingelheim), 10-30mM in DMSO
reagents for cloning and PCR
cell type of interest
cell culture media
2x SDS lysis buffer
PBS or HBSS
sonicator with microtip, or 25U/μL benzonase nuclease (Novagen)
SDS-PAGE and immunoblotting equipment and reagents
rat 2F2 (Roche) or mouse 12CA5 (Roche) or mouse HA-7 (Sigma) or chicken anti-HA (ICL) antibody
mouse anti-T7 antibody (Novagen)
secondary antibodies for detection by chemiluminescence or fluorescence chemiluminescence substrate (if performing chemiluminescence)
autoradiography film (for chemiluminescence) or immunoblot imaging system (for chemiluminescence and fluorescence)
Construct a TimeSTAMP fusion to the gene of interest
1. Identify whether the protein of interest is best tagged at its N-terminus or C-terminus.
A terminus that is unlikely to interfere with the activity or localization of the protein should be selected. (See “Critical Parameters.”)
2. Obtain an expression plasmid for the protein of interest.
Depending on the nature of the experiment, the promoter could be a native promoter (such as that of the gene for the protein of interest), a constitutive viral promoter (such as CMV), or an artificial promoter (such as a tet-responsive promoter). Other elements should be included depending on the nature of the experiment, e.g. introns or untranslated regions if the investigator wishes to reconstitute regulation of mRNA splicing or transport.
3. Select for fusion either the TimeSTAMPa or TimeSTAMPt cassette, for analysis of proteins undergoing low or high rates of synthesis, respectively, or test both versions.
The TimeSTAMPa cassette is typically used for low abundance proteins or proteins with slow ongoing synthesis rates whereas the TimeSTAMPt is typically used for highly expressed proteins or proteins with faster ongoing synthesis rates. For proteins whose characteristics are not well understood, both versions can be tried in parallel. (See “Critical Parameters.”)
4. Subclone each complete TimeSTAMP cassette in frame to either the N- or C-terminus of the gene.
The complete cassette consists of N-terminal T7 epitope tag, N-terminal cleavage site, NS3 protease, C-terminal cleavage site, and C-terminal HA tag. In the original TimeSTAMP cassettes, SrfI and AscI restriction sites can be used to transfer the entire cassette, if SrfI and AscI sites are first inserted into the beginning or end of the coding region of the gene of interest. The proper translation frames reading through the SrfI and AscI sites are +2 and +1, where the first nucleotide of the +1 frame is the first nucleotide of the recognition site (). Alternative sites that do not exist in the sequence of the TimeSTAMP cassette (available from the authors) can also be used, in which case the TimeSTAMP cassette can be amplified with PCR with primers containing these sites in place of SrfI and AscI.
5. Verify that the cloning junctions and TimeSTAMP sequences are free of unwanted mutations by sequencing, and prepare plasmid DNA for transfection by anion-exchange chromatography.
Commercial anion-exchange chromatography “maxiprep” kits such as those available from Qiagen or Invitrogen are preferred. Silica adsorption chromatography as found in most “miniprep” kits and some “maxiprep” kits do not yield as consistent results in transfections.
Verify proper functioning of the TimeSTAMP cassette
6. Grow a transfectable cell type to the proper density for transfection in a 12-well plate. Prepare two wells for each construct to be tested, plus one well to serve as an untransfected control.
For instance, HEK293 or 293T cells are reliably transfectable at 80-90% confluence by commercial liposomal reagents such as Lipofectamine 2000 (Invitrogen) or Fugene (Roche), or by calcium phosphate. Two wells are required per construct, as one well will be treated with BILN-2061 and the other left untreated.
7. For each construct to be tested, transfect the two allocated wells with 0.8 μg DNA each with a liposomal reagent or with calcium phosphate.
Perform liposomal or calcium phosphate transfections according to standard protocols, except use only 0.8 μg of DNA to avoid toxicity, adjusting all other volumes in the transfection mixture accordingly.
8. For each construct being tested, prewarm two tubes each of 1 mL of standard culture media with serum. To one tube, add BILN-2061 to a final concentration of 3 μM. Two hours after addition of transfection reagent to cells, for each construct, remove the media in the two wells. To one well, add the 1mL of media without BILN-2061, while to the other well, add the media without BILN-2061. Return the plate to the incubator for 24 hours.
If HEK293 or 293T cells are being used, apply new solutions slowly to the wells in order to prevent detaching the cells from the surface.
9. For each construct, prepare 500 μL 2x reducing SDS lysis buffer by adding 2-mercaptoethanol to 2x SDS lysis buffer to a final volume of 10%, then heating to 85-95° C in a chemical safety flow hood. Prepare for each well 1 mL of wash buffer in the form of PBS or HBSS.
A hot solution of SDS and 2-mercaptoethanol, rather than a non-ionic detergent solution, is used to lyse the cells, as this solution causes immediate denaturation of cellular proteins. Destruction of the protease activity within the TimeSTAMP cassette allows the omission of BILN-2061 from the lysis buffer.
10. Remove the transfected cells from the incubator, then, working quickly, replace the media in each well with 1mL wash buffer, dispensing the wash buffer gently onto the wall of the well so as not to wash the cells off the surface. After accomplishing this for all wells, quickly aspirate away the wash buffer. Immediately move the plate to the hood, then quickly dispense into the center of each well 200 μL of the 2x reducing SDS lysis buffer. After lysis buffer has been dispensed into all the wells, agitate the plate to spread the lysis buffer around the entire surface of the wells.
The purpose of the wash step is to remove serum components, primarily albumin, whose presence in the lysate causes anomalous migration during electrophoresis and ghosting during immunoblotting. It is important to work quickly from the time wash buffer is applied to the cells until it is removed. The wash buffer does not contain BILN-2061, so BILN-2061 will begin to diffuse out of cells during the wash. This is not a concern as long as the wash is less than 1 minute in duration, as a negligible proportion of the complex of NS3 and BILN-2061 will dissociate in this time (Flores et al., 2009
11. With the plate tilted, scrape down the lysate, which will be viscous, to the bottom of each well, then transfer into a 1.5mL microcentrifuge tube and place on ice.
The wide end of a 1mL pipette works well for scraping down the lysate.
12. Shear the DNA in each sample by sonication with a microtip sonicator. Alternatively, add 1 μL of 25U/μL benzonase nuclease and Mg2+ to 1 mM final concentration to each sample, mix well, and incubate at room temperature for 30 minutes.
Sonication is faster but requires the availability of a sonicator and familiarity with its use. Benzonase is a convenient substitute for laboratories without a sonicator, but requires purchase of the enzyme.
In Branson or Misonix sonicators, 5 s with the power set to the microtip limit should be sufficient. Switch the power on and off while the tip is immersed in the sample to prevent foaming.
Addition of 1 mM Mg2+ enhances benzonase activity. Check that the sample is no longer viscous by pipetting with a 200 μL pipette tip or flicking the tube. Repeat sonication or benzonase digestion if viscosity persists.
13. Heat the samples again on a heat block at 85-95° C for 2 minutes. Mix and centrifuge quickly in a microcentrifuge to collect the sample at the bottom of the tube.
At this point, the lysates can be stored at −20° C for analysis by immunoblotting at another time.
14. Perform SDS-PAGE with 2-4 μL of each sample in duplicate, followed by immunoblotting, according to standard protocols. For one set of lysates, perform immunoblotting with anti-T7 antibody. For the other set, perform immunoblotting with anti-HA antibody. Use any immunodetection method of choice.
Analyze the immunoblots to determine if the TimeSTAMP cassette is being efficiently removed in the absence of drug, and completely retained in the presence of drug, and that no unexpected cleavage occurs in the protein of interest. For fusion proteins with the TimeSTAMP cassette at the C-terminus, the T7 epitope detects protein produced both in the absence and presence of BILN-2061. Proteins produced in the presence of drug should not have undergone TimeSTAMP cassette removal, and should appear 30kD larger than proteins produced in the absence of drug when probed with anti-T7. The untransfected well serves to identify which bands identified by the anti-T7 antibody are non-specific background bands. The HA epitope, in contrast, is removed by protease activity, and therefore is present only on proteins produced in the presence of BILN-2061. Anti-HA protein should only detect the full-length uncleaved protein in the lysate exposed to BILN-2061. For proteins bearing the TimeSTAMP cassette at the N-terminus, the roles of the T7 and HA epitopes are reversed.
Quantify target protein synthesis rates over defined times with TimeSTAMP
15. To study protein production rates over a defined time window with treatments of interest, repeat steps 6-7 above, calculating how many wells are needed for all experimental conditions (which may be a combination of treatments to be tested and time windows in which protein synthesis will be observed).
For instance, if cells are to be either untreated and treated with a growth factor and protein synthesis over two different time windows analyzed, then at least seven wells are necessary: two time points with BILN-2061 multiplied by two treatment conditions, plus one well as a transfected but untreated and uninhibited control, a transfected well that is treated continuously with BILN-2061, and an untransfected well.
16. Following transfection for two hours, move the cells into media. For the control well treated continuously with BILN-2061, include the BILN-2061 at that time. For other wells, add the BILN-2061 later at the desired time points. Perform experimental treatments as desired.
If a well is to be cultured for longer than two days following BILN-2061 addition, refresh BILN-2061 every two days by removing 0.5 mL of the culture media with old BILN-2061 and replacing with 0.5 mL of culture media with fresh 3μM BILN-2061. When adding BILN-2061 at later time points, it is best to make a 30μM solution of BILN-2061 in media to be used as a 10x mix instead of adding BILN-2061 directly from the DMSO stock onto the cells. Direct application of the stock DMSO-based solution of BILN-2061 can result in DMSO-mediated toxicity in regions of high DMSO concentrations and uneven dispersion of BILN-2061.
17. Detect newly synthesized proteins following BILN-2061 addition by performing steps 9-14 above.
If utilizing chemiluminescence or fluorescence for antibody detection in the immunoblot, the relative quantities of old and new proteins in each sample can be calculated from the emission counts collected from the faster and slower migrating bands, respectively, detected by the constitutive tag (T7 for TimeSTAMP fusions to the C-terminus of the protein of interest, HA for fusions to the N-terminus).
Support Protocol: Immunocytochemical detection of newly synthesized proteins by drug control of TimeSTAMP tags
Immunocytochemistry allows visualization of the spatial distributions of newly synthesized proteins of interest.
TimeSTAMP fusion construct (see Basic Protocol)
12mm coverslips and 24-well plate, or 35mm glass-bottom tissue-culture dishes (Mattek)
cell type of interest
cell culture media
BILN-2061 (Boehringer Ingelheim), 10-30 mM in DMSO
PBS with 0.1% Triton X-100
8% paraformaldehyde in PBS, pH 7.2-7.5 (see Reagents and Solutions)
Blocking solution (see Reagents and Solutions)
rat 2F2 anti-HA (Roche)
mouse anti-T7 antibody (Novagen)
rabbit or chicken antibody to protein of interest (if available)
Alexa Fluor 488-conjugated goat anti-mouse IgG, highly cross-absorbed to rat serum proteins (Invitrogen)
Alexa Fluor 568-conjugated goat anti-rat IgG, highly cross-absorbed to mouse serum proteins (Invitrogen)
Alexa Fluor 647-conjugated goat anti-rabbit or anti-chicken IgG (Invitrogen)
Vectashield mounting solution (Vector Laboratories)
Fluorescence microscope with filters for fluorescein, rhodamine/Texas Red, and Cy5 wavelengths.
Donkey secondary antibodies can be used instead of goat secondary antibodies if the experimenter desires to use a goat primary antibody to the protein of interest.
Transfect cells with TimeSTAMP reporters
1. For cell types to be transfected in adherent monolayers by liposomal reagents or calcium phosphate, grow a transfectable cell type to the proper density for transfection on 12 mm coverslips in a 24-well plate or on 35 mm glass-bottom tissue-culture dishes, using enough wells or dishes for the combinations of time points and treatment conditions desired. One well or dish should be reserved for a no-drug control control. For cell types to be transfected by electroporation (e.g. using the Amaxa Nucleofector protocol), prepare the coverslips or plates separately and obtain the proper number of cells for each transfection condition according to the electroporation protocol for the desired cell type.
2. Transfect with the reporter plasmid expressing the protein of interest fused to a TimeSTAMP cassette, whose responsiveness to drug was previously verified by immunoblotting (Basic Protocol). Any time after transfection is complete (e.g. 2 hours for adherent cells, or after electroporated cells have adhered), but prior to performing drug administration, transfection media should be replaced with fresh media to remove transfection reagents or dead cells.
3. Perform biological treatments (e.g. growth factor treatment if investigating the effects of a growth factor on synthesis of a protein of interest) as desired. If the experimenter desired to perform a time course of treatment, e.g. 1, 3, 6, 12, and 24 hours, it is convenient to perform the treatment on different wells at those time intervals prior to a common experimental end time. Add BILN-2061 to begin the time interval in which newly synthesized proteins are to be tagged. Typically, this is immediately after the biological treatment or stimulation, but it is also possible to delay the beginning of the tag preservation window relative to the treatment time.
It is best to make a 30μM solution of BILN-2061 in media to be used as a 10x mix instead of adding BILN-2061 directly from the DMSO stock onto the cells.
4. At the end of the time window in which newly synthesized proteins are to be visualized, add 1 volume of 8% paraformaldehyde, equilibrated to room temperature. Incubate at room temperature for 15 min.
Adding paraformaldehyde to the media with BILN-2061 achieves both fixation and destruction of protease activity without allowing escape from inhibition.
5. Rinse with PBS 3 times for 3 minutes each, then permeabilize and block with blocking solution for 30-60 minutes at room temperature.
6. Prepare primary antibody solution by diluting rat 2F2, mouse anti-T7, and rabbit antibody to protein of interest (if available) to 1 μg/mL each in blocking solution. Incubate cells in primary antibody solution at room temperature for 1-2 hours or at 4° C for 12-24 hours.
7. Prepare secondary antibody solution by diluting Alexa Fluor 488-conjugated anti-mouse IgG, Alexa Fluor 568-conjugated anti-rat IgG, and Alexa Fluor 647-conjugated anti-rabbit IgG to 0.5 μg/mL each. Remove primary antibody solution from cells, rinse cells with PBS with 0.1% Triton X-100 3 times for 3 minutes each, then incubate cells in secondary antibody solution at room temperature for 30-60 minutes.
Secondary antibody solution can be prepared in PBS with 0.1% Triton X-100 without normal goat serum as well.
8. Wash with PBS with 0.1% Triton X-100 3 times for 10-15 minutes each. Remove final wash by aspiration. For coverslips, invert onto a 3 μL drop of Vectashield on a glass side, aspirate away excess Vectashield from the coverslip edges, and seal the edges with fingernail polish. For glass-bottom dishes, cover the glass area with Vectashield, replace the lid on the dish, and seal with wax film to prevent evaporation. Samples can now be stored at 4° C for up to a week or imaged on a microscope.
Here, Alexa Fluor 488 is used to detect the N-terminal T7 tag while Alexa Fluor 568 detects the C-terminal HA tag. If the TimeSTAMP cassette is at the N-terminus of the protein of interest, Alexa Fluor 488 fluorescence would reveal newly synthesized protein copies while Alexa Fluor 568 fluorescence would reveal all protein copies. If the TimeSTAMP cassette is at the C-terminus, the opposite relationship holds. Signal from the Alexa Fluor 647-conjugated anti-rabbit or anti-chicken secondary antibody (invisible to the human eye) can be used to compare distributions and levels of endogenous and transfected protein of interest, if a specific antibody is available. These are suggested wavelengths; researchers may rearrange the wavelengths used for their convenience. Alexa Fluor 555 may be used in place of Alexa Fluor 568, but in our experience is dimmer and slightly more prone to aggregation. Note anti-T7 antibodies typically show some cross-reactivity to cell nuclei.