Agilent 1260 is new system and model.
AU: 3-28-2016
HPLC Questions- AU-QUESTIONS TO PONDER.docx
RC: I need the following clarifications asap:
"1. Beckman has a Model 166 UV-vis detector with a dual photodiode sensor, whereas both Agilent models have a photo-diode array detector."
- As previously discussed, you should provide commentary on how this affects detection sensitivity. Practically speaking, can we accurately quantify
smaller peak areas. Would we expect better S/N.
AU: Having DAD detector is useful because it can detect at least two wavelengths simultaneously whereas UV can detect only one at a time. Using DAD, we can set for multiple wavelengths to detect different compounds if they absorb different wavelength. This way quantification confidence is increased.
RC: Still need commentary on sensitivity.
AU: 3-30-2016: Sensitivity of detection depends upon collectively on the choice of the detector, choice of column (binding capability) and the method used (efficiency of separation). This was described, to some extent, in following paper. hplc sensitivity.pdf
SM: There is no Integration function in the conventional UV-Vis spectrophotometer which accumulates the signal. For example, the total required time will be 1000 sec for 1000 data points and it takes 1 sec to measure one data point. In this case, all 1000 data points have the same signal to noise ratio (S/N). But in a PDA instrument which has a 1000 photodiode arrays, 1000 data points can be measured in 1 sec and it would take 1/1000 sec to achieve the same result obtainable in 1 sec in a conventional instrument. Therefore, when the same sample is measured for 1000 sec in a PDA instrument, the signal is accumulated and is 1000 times greater than when measuring for 1 sec. The noise will be sqrt(1000) . This means that the S/N ratio is improved by sqrt(1000).
"The 32-Karat software used with the Beckman system has limited data acquisition and analysis capabilities, whereas the Chemstation software used with the Agilent systems have advanced data acquisition and analysis features."
- Such as?
AU: 32 Karat is really old software where as Chemstation is new wich has better flexibilities.
SM: For example, creating, running and modifying methods is simple using Chemstation. It also has a better GUI with a lot of parameters and data displayed at the same time. In addition, customized reports can be saved and exported in various formats such as .doc and .pdf. Chemstation also has an offline mode that can be used simultaneously with the online mode (used for running samples), for data analysis. Most importantly, the raw data (analyzed or not) from Chemstation can be exported in formats such .csv to create publication quality figures.
"-In-line degasser
- system with in-line fraction collector which can be controlled by the in built ChemStation software."
-You mentioned it comes with degasser. Are these add-ons at extra cost or do you mean they are included?
AU: Degasser is in-line, comes with the system, Fraction collector with extra cost. I think we dropped the idea of buying it.
"I think in the new HPLC they have Quaternary pump and they are also giving C18 column."
-Please specify the advantages of quaternary pump.
AU: I think C18 column comes with the system and as of now we do not have any practical advantage of having Quaternary pump. My comment was why they have higher price so I speculated that may be they are giving column, and with quaternary pump, the price is high.
SM: The quaternary is a low pressure mixing pump whereas the binary is a high pressure mixing pump. The optimum gradient range for the quaternary pump is between 5% to 95% organic, whereas the binary pump shows optimum performance from 0% to 100% organic (which is what we use), which is advantageous for compounds eluting at very low or at very high organic percentage. The quaternary pump has the advantage that ternary and quaternary gradients can be applied (not applicable for us).
Another advantage for the binary pump is the high mixing efficiency, which is necessary if TFA is used as a modifier (applicable for us because we use TFA also).
Thus, for our needs, the binary pump is a better choice.
"ü Better maintenance and technical services"
RC: Please clarify differences in maintenance/tech support for the two Agilent products.
AU: I will look carefully tomorrow and update on this.
Meanwhile, Sudipto and XG can further comment on these specific points.
3.28.2016
XG: HPLC notes
QUESTIONS TO PONDER_XG (2).docx
SM: HPLC notes
QUESTIONS TO PONDER_Answered_SM.docx
Comparison between Beckman System Gold HPLC and Agilent.docx
Agilent 1100 Series spreadsht comparison_Answered_SM.docx
3.28.2016
Gertz and Steegborn 2016 review mention DHPs (also honokiol), indicating DHP-dependent activation of SIRT2 and 3 have not yet
been confirmed with non-FdL substrates. Note Steegborn was an author on the Dec 2015 DHP paper in JMC. He did not cite that paper
since it appeared after this review was submitted, but he seems to have excluded SIRT1 from this statement because the 2015 JMC paper
studied SIRT1 with another fluorophore based assay that does not use a tagged substrate (Sirtainty).
Regarding FdL, he speculates that DHPs may interact with peptide substrate, but indicates that mechanistic characterization is pending.
Note the importance of use of saturating [peptide] in our experiments in this regard.
It is possible that Steegborn is currently investigating DHP activation in more detail.
3.23.2016
Two new experiments specified below. Please provide schedule for these starting wed.
- compounds with intrinsic fluorescence should be dealt with carefully in fluorescence based screens/assays
- problem is even with standard curves, depending on standard used baseline subtraction of the standard may not be valid.
- we were prohibited from running a rigorous standard curve because we do not know whether the FdL standard is identical to deacetylated FdL peptide and do not have deacetylated FdL peptide. From our previous discussions it is not entirely clear why the FdL standard should be different, but they said it is proprietary or did not answer. If you have any other info about this please let me know.
- as we discussed we could synthesize deacetylated FdL peptide and use that to run proper standard curves. We also discussed other approaches that would be more costly
1) for now we should immediately follow this simpler approach, which provides us with a proper control for purpose of determination of whether the increase in FdL signal in presence of dhp can be interpreted as activation:
For each of the dhps we have worked with in the past (dhp 1c, 2c and modified 1b), do the following
Run the reaction (eg at 500uM NAD) without Dhp
Add dhp after the reaction
Quantify afu
Then compare afu from above reaction to a parallel reaction that differs only in that dhp was added before reaction (as you usually do)
Finally determine whether reaction with dhp added before reaction gives higher afu than that where dhp was added after reaction. Higher afu may be interpreted as more deacetylated product formed with dhp present during reaction, though this approach is not to be used as replacement for proper standard curve which is needed to determine how much product
Ideally some of the dhps can done together. If not 2c is priority followed by 1c followed by 1b derivative
- you can use any Enzo batch available for this.
Please do this as a priority while alok runs the planned reactions with Hplc analysis (no DMSO, 500uM NAD). We need these results fast. Please tell alok to plan to start those expts on wed or th
Later you can repeat the same with sirt1
- I trust you are using saturating peptide concentration (we previously used around 250 for saturating I believe) for all these expts based on my comments on wiki on mon
XG: The Km(FdL peptide) =32 uM.
- Related, the new jmc paper with sirtainty also uses fluorescence and does not avoid issue w intrinsic fluorescence.
If you have information about the standard for sirtainty (eg is it proprietary or disclosed to be identical to product produced in assay) let me know
2) Please also immediately verify whether we have honokiol in the lab.
The immediate next step for you after the above will be:
move on to honokiol, which likely does not have intrinsic fluorescence and has preliminary evidence suggesting nad km reduction as mode of activation. I believe we have
it in lab. Please starting wed then immediately move on to endpoint assays of honokiol at different nad concentrations. Check whether the activation stops above a certain nad concentration. Then, we could move on to do the various initial rate assays as planned but now with this compound
3.21.2016
-- As discussed experiments 1) on NAM dose-response (comparing HPLC and FdL), and 2) the lower [NAD+] endpoint DHP activation study comparing HPLC and FdL will be the priorities.
AU (HPLC): NAM dose response with following concentrations-
[NAM] = 0, 10, 50, 100, 200 uM
[NAD+] = 500 uM
[FdL peptide] = 100 uM
Enzo Sirt3 = 1 uL
37 degree, for 1hrs
XG (FdL assay): NAM dose response with following concentrations-
[NAM] = 0, 10, 50, 100, 200 uM
[NAD+] = 500 uM
[FdL peptide] = 100 uM
Enzo Sirt3 = 1 uL
37 degree, for 1hrs
XG (FdL assay): DHP2c activation-
[DHP2c] = 0 and 75 uM
[NAD+] = 0.5 mM and 1.5 mM
[FdL Peptide] = 100 uM
Enzo Sirt3 = 1 uL
37 degree, for 1hrs
RC: I assume 100uM peptide is close to saturating for this peptide. This will certainly be important for the experimental data we put in the paper.
-- Based on Alok's comments below it seems your involvement on the FdL experiments may be warranted.
-- Since we may want to confirm activation of this batch using FdL before committing large amounts of time to the corresponding HPLC analysis, it may make sense for you
to do the FdL experiments for both 1,2) while Alok does 1) NAM dose-response with HPLC.
-- Then depending on
-the extent of activation by DHP we measure with FdL at [NAD+]=500uM and 1500uM, and
-confirmation of consistent inhibition by NAM using FdL and HPLC measurements,
we can decide whether to proceed with HPLC analysis for lower [NAD+] without any further modifications, or whether other experiments are needed.
-- Need to decide whether to use:
-1 hr or 2 hrs. Unless S/N is limiting, 1 hr is generally preferred.
AU: We are going to do 1hrs reaction time. I have enough enzyme to complete the experiment, both me and XG.
-For 1), 500 or 1500uM NAD+. 500uM NAD+ is generally preferred (1500uM would help show consistency of FdL/HPLC under the same conditions as Fri's DHP 2c activation HPLC experiment, but since we are altering the conditions going forward anyway, that is not critical).
2) would use both 500 and 1500uM NAD+ for FdL. It would use 500uM NAD+ for HPLC.
-5% DMSO or no DMSO. No DMSO is generally preferred. Certainly no DMSO will be used for 1). For 2), you can decide based on your extent of concern regarding DHP solubility.
No DMSO for both the experiments.
-what enzyme concentration for HPLC. As I understand this will be the only point of difference between FdL and DMSO. Please confirm
and specify the choice for detectable peak area.
For both HPLC and FdL assay, we are planning to use 1 uL Sirt3.
RC (3/21): Regarding Wed, I wanted to make sure you are not idle waiting for me to revert in afternoon.
Here is what I propose:
-- Provide bullet point summary of results by Wed noon. If report is ready, provide by then.
-- If needed complete report in afternoon.
-- If report is complete by midday Wed, proceed as follows in afternoon unless I revert to you regarding feedback on the results/next steps:
a) XG: continue with steps needed for urea purification (and/or completion of any other data sharing tasks outlined today).b) AU: run the NAM peak area / concentration correlation expts we discussed last week (for purpose of using NAM peak for product quantification in future).AU: 3-21-2016- Yes, thank you
3.20.2016
It is essential that RC receives further information on experimental protocols immediately. Both of you should address these as a priority on Mon before doing anything else.
a) Reviewing the dose-response protocols for AU reveals use of 1500uM NAD+ and 2 hr reaction time
Reviewing biorxiv and earlier reports of XG reveals use of 500uM NAD+ and 1 hr reaction time for EC1.5/dose-response expts. (PLOS reports some endpoint expts using 100uM NAD+.) On the other hand, I see a table on wiki that claims 1500uM NAD+ was used for EC1.5 experiments with DHP 1c. Reviewing all prior data on wiki and email, I could not find any indication of this value prior to the posting of this table, or an explanation as to why it was used. so far I could only find one comment about [NAD+] in FdL endpoint studies -- on an email from 12/15/15 there was mention of high [NAD+], but nothing quantitative.
Why the difference (1500 vs 500uM NAD+)? Low activity of urea enzyme? A physiological concentration of NAD+ was stated in biorxiv to be 375uM, which could have justified use of ~500uM NAD+.
XG: DHP1c EC1.5 measurement was firstly repeated in the lab by using original reported condition (2009 JMC paper). In that paper, “250 μM substrate, and 500 μM NAD+ at 37oC with 5U Enzo SIRT3” were the conditions. Data reported in biorxiv was using this condition.
To save the reagents, DHP1c EC1.5 for SIRT3 was measured under following condition “100uM substrate, 500uM NAD+ at 37oC with 2.5U Enzo SIRT3”. The results were comparable.
When we test in house enzyme, condition of 1500uM NAD+, 100uM substrate at 37oC was used. And this condition was used all the time thereafter for both Enzo enzyme and in –house enzyme.
Since I don't have access to that info I cannot complete the analysis and am compromised in ability to review when you are not in lab.
(Note: The biorxiv also says [peptide]=250uM whereas the wiki table says 100uM. Again not clear why the difference. though this may be less critical to the current analysis.)
b) RC had asked for time series of product formation vs time from FdL initial rate experiments w/, w/o DHP for the purpose of assessing progress of the reaction and appropriate choice of endpoint time. All concentration info for the protocol should be included. I would like to see the curves for all the different [NAD+] used in the initial rate experiments. I could not find these on wiki after searching myself. This can be provided for DHP 1c and/or 2c (ideally both if available).
Also, cross reference these against the corresponding endpoint data -- namely, the EC1.5 experiments that were used to choose [DHP] for the initial rate experiments.
XG: Data of time series of product formation vs time from FdL initial rate experiments with/without DHP1c has been posted on wiki.
3/11/16
Discussion on SIRT3 purification protocol from : Crystal Structures of Human SIRT3 Displaying Substrate-induced Conformational Changes. JBC. 284,(2009) 24394–24405
The author’s describe purification protocols for two clones of human SIRT3 (102-399 and 118-399), both of which are purified from the soluble fraction to >95% purity.
- The author’s make no mention of the yields for the purified proteins.
- The purification procedure was tried with SIRT3 in our lab previously, but without success. Soluble protein was not being produced.
- The 102-399 clone was co-expressed with a chaperone protein (pG-KJE8), however the author’s do not mention if they had any issues with co-elution of the chaperone protein with SIRT3.
- The author’s used nM quantities of purified enzyme to drive reactions containing saturating amounts of substrate, suggesting that the enzymes are highly active.
- The activities of the two clones were found to be very similar by these authors, however the 2008 JMB paper (Schlicker et al) suggested that SIRT3-(114–399) has a ~ 50-fold-higher specific activity than SIRT3-(102–399). The cause of this discrepancy is not addressed.
Differences in the implementation of the protocol: Differences are highlighted in red.
RC: Please provide some commentary on the differences, why these differences occurred, possible effects, etc so we can determine whether they could be reasons for the varying results and whether there are any questions to ask the authors. You can do this between expts; don't slow down.
JBC:
- Human SIRT3-(102–399) was cloned into a modified pET21b vector (Novagen) and expressed in BL21(DE3) cells (Novagen) with co-expression of chaperone pG-KJE8 (Takara).
- A single colony was inoculated in LB media containing 20 g/ml chloramphenicol and 100g/ml ampicillin for plasmid selection and 5 ng/ml tetracycline and 0.5 mg/ml L-arabinose for chaperone induction at 37 °C, 250 rpm until the A600 reached 0.3.
- The culture was then transferred to 16 °C, 250 rpm until the A600 reached 0.6. isopropyl 1-thio--D-galactopyranoside was added to a final concentration of 0.3 mM, and expression was continued at 16 °C, 180 rpm overnight.
- Cells were collected by centrifugation, and the pellet was resuspended in lysis buffer (10 mMimidazole, 300mMNaCl, 10% glycerol, 50mMTris-HCl,pH 7.8, 10 ml/gm of cell pellet) and sonicated to open the cells.
- Supernatant was separated from cell debris by centrifugation at 11,000g for 40 min at 4 °C and loaded onto a Ni-NTA column (Qiagen).
- The column was washed with 30 column volumes of the lysis buffer and 20 column volumes of the buffer containing 20mM imidazole, 300mM NaCl, 10% glycerol, 50mM Tris-HCl, pH 7.8, and eluted with the buffer containing 100 mM imidazole, 300 mM NaCl, 10% glycerol, 50 mM Tris-HCl, pH 7.8.
In-house:
- Human SIRT3-(102–399) was cloned into a pEX-N-His vector and the cloned plasmid construct was purchased from OriGene. The plasmid was transformed into BL21 cells.
- A single colony was inoculated in LB media containing 100 g/ml ampicillin at 37 °C, 250 rpm until the A600 reached 0.3. The culture was then transferred to 18 °C, 250 rpm until the A600 reached 0.6–0.8.
- Isopropyl 1-thio--D-galactopyranoside was added to a final concentration of 0.3 mM, and expression was continued at 18 °C, 160 rpm overnight.
- Cells were collected by centrifugation, and the pellet was resuspended in lysis buffer (200 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, and 25mM HEPES-NaOH, pH 7.5) and sonicated to open the cells.
- Supernatant was separated from cell debris by centrifugation at 10,000 g for 40 min at 4 °C and loaded onto a His GraviTrap column (GE Healthcare) that equilibrated with the buffer containing 200 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, 20 mM imidazole, and 25 mM HEPES-NaOH, pH 7.5.
- The column was washed with 5 column volumes of the buffer containing 200mM NaCl, 5% glycerol, 5mM 2-mercaptoethanol, 50 mM imidazole, and 25 mM HEPES-NaOH, pH 7.5, and eluted with the buffer containing 200 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, 250 mM imidazole, and 25 mM HEPES-NaOH, pH 7.5.
RC: Some additional comments about the subdenaturing urea article:
-- they seem to reference denatured bacterial lysate protocol among other prior approaches to removal of Cpn from AE purified proteins
-- they indicate that a previously reported method using ATP/Mg did not work for their protein. May not have used the combined ATP/Mg and denatured
bacterial lysate approach
-- they tested on two proteins; for the other, they apparently removed most of Cpn, but purity was lower.
-- mechanism involved partial denaturation or conformational change of Cpn causing it to dissociate from target proteins
-- it is indicated with reference to a prior article that without Cpn removal, the soluble aggregates can have altered kinetic and structural properties (meaning removal of Cpn is important for
mechanistic studies)
-- 1M urea was sufficient to remove 95% of Cpn band in case of the enzyme purification; 2M achieved ~100% of Cpn band removal. If there are any concerns about partial denaturation
of target protein, may not need to get 100% removal.
-- specific activity of the highest purity wash below is also highest, but not always in proportion to the increase in purity
-- nonetheless it is interesting that high purity (with respect to Cpn band) is associated with high specific activity for this method
-- I assume we will be optimizing [urea] in wash 2 buffer for SIRT3
SM: Yes, I will first try the recommended 2M urea in the wash buffer and see what kind of purity I get. Based on those results I will try to optimize the wash step for highest purity.
Given the fact that removal of Cpn from SIRT3 is essential for both: a) accurate mechanistic studies b) increase in specific activity, in case the subdenaturing urea method does not work, I could try a combination of approaches such as combining the Mg/ATP + denatured lysate method with the subdenaturing urea method.
RC: Ok so 2M urea is supposed to disrupt Cpn60 - enzyme interactions without decreasing enzyme activity significantly (little denaturation).
This was shown for at least one other protein in the literature, as I understand. Did they report enzyme activity with / without treatment?
AU: Please see table 1 (attached: Table 1-Belval et al)
RC:“Please elaborate on whether total activity is increasing with 2M urea treatment and why”
AU: Looking at the table, it appears that the total activity is increasing with 2M urea treatment/wash. This is also true with wash 2 which doesn’t have urea. The only mention I see, regarding activity, is this-
“Total activity was highest following purification with wash buffers containing urea (Table 1, lines 3, 4 and 5), whereas LEW 1_ and Tris/imidazole (Table 1, lines 1 and 2) led to a loss of around 10–15% of total activity.”
AU: There are many unanswered questions in this table. E.g. from 3-5, all have 2M urea and this paper says Urea is major contributor (my interpretation is that in this method, ATP/Mg should not interfere in purity) then why purity in these decreased? Moreover, #2 (no urea) has better purity than #4 (with urea). And authors do not discuss these in their paper.
RC:I assume denatured bacterial lysate will take more time to prepare and that the urea approach seems to be more efficient (given your comment about the lower quantity of impurity in method 2 paper).
AU: I think the urea method is more straightforward, although some optimization will be needed in both the cases. In case of urea method, they mention that the Cpn60 impurity was 55% (similar to our case), whereas in denatured bacterial lysate paper, I visually compared the amount of Hsp70 in different lanes and to me it appeared less as compared to ours.
RC: Is anything known about activity effect of the suboptimal urea treatment?
AU: In their study (Table 1; Belval et al., 2015), the specific activity was highest with 2M urea in buffer.
Protocols to remove Cpn60 - Arctic Express cells.docx
RC: Please see these tables from Guan:
| 1mM IPTG induction at 30 oC |
1M Urea (week of 10.16.2015) |
4M Urea (week of 10.16.2015) |
6M Urea (week of 10.16.2015) |
8M Urea (week of 10.16.2015) |
| [Purified Protein], ug/ul |
0.1451 |
0.1863 |
0.2725 |
0.5196 |
| VTotal, ul |
2500 |
2500 |
2500 |
2500 |
| Total protein, ug |
362.8 |
465.8 |
681.3 |
1299.0 |
| Specific activity, U/ug |
0.0452 |
0.0964 |
0.4453 |
0.4052 |
AU: I think it is combination of both recovery of the protein and purity combined. XG, please comment on the purity of the preparation.
XG: The yield and purity were low compare to those using 6/8M urea.
RC: Please quantify low purity as much as possible and
XG: The yield was too low to quantify (please see the attached ppt).
RC: Also, it's interesting to note from the following table:
| 1mM IPTG induction at 30 oC, 8M Urea treatment |
Batch I_ 800mL (week of 10.26.2015) |
Batch II_600mL (week of 10.26.2015) |
Batch III_800mL (week of 11.02.2015) |
Combined Batch I/II/III |
Arctic express cell 3mM IPTG, 0.5% NP40 |
| [Purified Protein], ug/ul |
0.3706 |
0.4259 |
0.6012 |
0.4828 |
0.9722 |
| VTotal, ul |
6150 |
4350 |
7000 |
14200 |
800 |
| Total protein, ug |
2279.1 |
1852.7 |
4208.4 |
6855.7 |
777.8 |
| Specific activity, U/ug |
0.4862 |
0.4289 |
0.4953 |
0.3987 |
0.9787 |
AU: certainly possible because, in AE, there is no denaturation.
RC: assume these specific activities were all based on the same Enzo batch for comparison.
AU: Yes. XG please confirm.
XG: I agree.
RC: What about 8M urea as reported in the literature: Was it shown in many cases to reduce enzyme activity / denature significantly? Is there any data on this for any proteins? I had asked this question earlier I believe.
AU: I believe the original protocol XG followed was for Sirt1 purification (or Sirt2, not sure). XG can elaborate on activity aspect of this enzyme.
XG: The original reference was for SIRT1 purification. There were cases to indicate denaturation of enzyme after Urea treatment.
https://www.agilent.com/cs/library/applications/5990-8141EN.pdf
RC:provide further elaboratedcommentary on 8M urea lit question (not just links).
XG: Please check the attached PNAS 2003 paper. The molecular basis for the chemical denaturation of proteins by urea_2003_Bennion.pdf
In brief, simulations of CI2 in 8 M urea indicate that urea promotes unfolding by both indirect and direct mechanisms. Direct urea interactions consisted of hydrogen bonding to the polar moieties of the protein, particularly peptide groups, leading to screening of intramolecular hydrogen bonds. Solvation of the hydrophobic core proceeded via the influx of water molecules, then urea. Urea also promoted protein unfolding in an indirect manner by altering water structure and dynamics, as also occurs on the introduction of nonpolar groups to water, thereby diminishing the hydrophobic effect and facilitating the exposure of the hydrophobic core residues.
RC:BTW, what is the meaning of "suboptimal" urea?
AU:Suboptimal concentration of urea means, the minimum amount of urea required to remove the Cpn60 from Sirt3 without significantly affecting the activity of the enzyme.
RC: I assume denatured bacterial lysate will take more time to prepare and that the urea approach seems to be more efficient (given your comment about the lower quantity of impurity in method 2 paper).
AU: I think the urea method is more straightforward, although some optimization will be needed in both the cases. In case of urea method, they mention that the Cpn60 impurity was 55% (similar to our case), whereas in denatured bacterial lysate paper, I visually compared the amount of Hsp70 in different lanes and to me it appeared less as compared to ours.
RC: Is anything known about activity effect of the suboptimal urea treatment?
AU: In their study (Table 1; Belval et al., 2015), the specific activity was highest with 2M urea in buffer.
RC (3.3.16): Please comment on sirtris paper on sirt3 since you said volumes of enzyme solution used are reported in literature (you didn't want to report high volume / low activity in your case). I believe you referred to this paper in this regard.
XG ( 3.3.16): In research article, the amount of protein used in the assay is required to be reported. Two ways normally used by researchers are (1) xx ug of protein was used in xx ul of total reaction volume; (2) xx uM of enzyme was used for the reaction. In Sirtris paper Biochemical characterization, localization, and tissue distribution of the longer form of mouse SIRT3, PROTEIN SCIENCE 2009 VOL 18:514—525, it was mentioned as following: “The partially purified full length and different fragments of mSIRT3L were tested for deacetylation activity with the mass spectrometry based assay reported previously.Saturating amounts of peptide substrate (20 uM) and bNAD+ (3 mM) were added to the reaction with enzyme concentration starting at 4 uM and serial diluted in a 1:2 ratio. Reactions were incubated at 25oC and stopped at 0, 15, 30, 60, 90, 120, 150, 180 minute time points with 10% formic acid with 50 mM nicotinamide. The conversion of substrate to product was determined by mass spectrometry in conjunction with a RapidFire system (BioTrove).”In our case, if we use 25ul of in house enzyme, by calculation it will be 15uM of enzyme used for reaction. For 40 ul, then 24uM. Instead of putting 25ul of enzyme, we say 15uM. It is not directly showing half of the total reaction volume is enzyme. As mentioned before, the assay system will be changed by addition of such much amount enzyme (in lysate buffer).
It’s also mentioned in the paper for inhibition analysis, mSIRT3L-54-334 (1.47nM) was used.
In terms of if 15uM or 24uM is too high, we have to look up more articles to get a general idea what is the common range of [protein]. In this paper, the purity of the enzyme was reported >95%.
RC (3.2.16): For low activity / high purity issue
XG(3.3.16)
ü Our first hand info was from Enzo. At that time, Enzo scientist (Jonathan Bohn <[email protected]>) responded according to low purity issue as following
“All lots of SIRT1 (BML-SE239) and SIRT3 (BML-SE270) are partially purified by single-step affinity chromatography and gel filtration. Please note these enzymes are purely sold by activity and not by weight. So, regardless of purity, there is still a defined amount of enzymatic activity. For most enzymes, activity degrades as a function of time. Therefore you will only have a finite period of time to manipulate the enzymes before the activity disappears completely. The severity of the drop depends on the molecule’s identity, but for some of them multi-stage chromatography is simply not an option. Since the activity is the most interesting aspect of the protein for most customers, these enzymes were developed to maximize specific activity, not purity.”
ü The gel picture shown less than 70% pure for the target protein (The target protein band should show below 36kDa marker).
ü The responses for couple of our questions is listed below
PMC-AT: Can you produce SIRT1/SIRT3 protein with higher purity such as >90%? If you can, we are willing to purchase. Would you provide the cost for such high purity? How long does it take?
Enzo: Our current purification method is intended primarily to service the needs of the related kit to provide maximal activity. It is entirely possible that we could further develop the purity specification, but in that case we can no longer guarantee the specific activitywe have seen historically. Active enzymes which are more extensively handled have a tendency to lose their enzymatic activity. If that’s acceptable, we would typically require fairly large bulk purchases or some other compensatory mechanism (eg, authorship or endorsement) to compensate for the cost of method development.
PMC-AT: Is that the case for human SIRT1(BML-SE239)? In short, lower or no activity when high purity is required.
Enzo: Yes. The impurities appear to be distinct, but at similar levels for BML-SE239 as well.
ü Literature* indicated that enzyme activity can be lost during purification steps depending the method/reagent used in the purification procedure.
*The problems associated with enzyme purification, E. Dako, AM. Bernier et al. Chemical Biology, chapter 2, 19-40. Edited by Prof. Deniz Ekinci.
RC(2.29.16): I also need some more info on how the specific activity of the same batch could change in hands of Sudipto vs Guan (the batch that Sudipto found had specific activity 0.18), and whether/how this depends on the Enzo batch that it is being compared to.
XG(2.29.16): The specific activity XG and Sudipto measured is relative specific activity. The steps are listed as following
(1) Certain amount of Enzo enzyme (2.5 U or 5U) will be used.
(2) Certain volume of in house enzyme (15ul or 20ul) will be used.
(3) At the same conditions ([NAD+], [Substrate], 60 min incubation, 37oC), the Enzo and in house enzyme will be applied for deacetylation reaction.
(4) Delta AFU was obtained and how many AFU produced by 1 U Enzo enzyme will be calculated.
(5) Then how many Unit produced by 15 or 20ul of in house enzyme will be calculated.
(6) The specific activity of in house enzyme will be then calculated based on concentration.
it does depend on how Enzo enzyme performs. Since there is variation from batch to batch for Enzo, the results can be difference.
RC: In that case, what if Sudipto used the old Enzo batch that you originally used for specific activity analysis, in order to assess the specific activity of his new batches?
What would the specific activities be in that case? Would they be much higher? Would some of them have specific activities comparable to your old batches?
In any case, we cannot report specific activities this way for the purpose of publication. If presented, they will need to be presented in a way that does not depend on Enzo.