ProteoCool Pills#25: Choice of the right material is essential to perform DNA and/or protein UV Spectrophotometric quantifications

 As already reported in the ProteoCool Pills#13, and ProteoCool Pills #24 several different methods are currently available to perform quantification of purified DNA fragments and plasmid as well as recombinant proteins and antibodies.

One simple method common to both, DNA and protein quantification is the spectrophotometric determination in the UV range (260nm for DNA and RNA and 280nm for proteins and monoclonal antibodies)

Spectrophotometric quantification has several advantages:

 - Cheap (do not require any specific reagent);

-  Fast (do not require sample pre-incubations);

- Non-destructive (the sample could be recovered);

The main drawback that limits in the past the use of the UV quantification was the fact that standard glass and standard plastic absorbs strongly in UV region and the quartz cuvettes were necessary to perform protein (280nm) and DNA (260nm) quantification.

Quarts cuvettes work well in both UV and visible regions (right from 190 nm) but are expensive, fragile and time consuming, because not disposable and therefore it need to be carefully washed between the different samples.

Of course the same limitation is applicable also to the multiplate reader, because the standard plastic bottom plates cannot be used for measurements in <300-320 nm due to the plastic absorbance.

Solutions:

1) Use a microvolume UV-Vis spectrophotometers (eg Nanodrop) that do not require any specific support (plate or cuvette):

  Pros:

 Low sample volume (2 µl)

 Fast

      Simple

       Cons:

          Less sensitive than cuvettes because the optical path is 1mm instead 10mM of the cuvettes

  Lambert-beer law à  Abs=ebc where b is the optical path

For the same sample e= constant à 1/10 of optical path à 1/10 of Abs at the same c (concentration)

 Need to be carefully cleaned (protein buffers are rich of salt and the surface properties of the pedestals can be compromised and the samples drop Flattens out and the read are not reproducible

       2) Use Plastic UV-Cuvette or  UVclear multiplates:

In the recent years special plastic compounds with low absorbance at wavelength >220nm were developed:

In this post I would like just to provide some example of comparision of background Abs260 and Abs280 signal obtained with standard and UV-transparent plastic matherial:

Semimicro BRAND 1.5ml PMMA cuvettes (Link)

BRAND UV-Cuvette SEMIMICRO

In conclusion, expecially for DNA determination, standard plastic cuvette and multiwell plates cannot be used. The UV transparent plastics represent a nice, cheaper alternative to quartz cuvette. If it is true that those support are little more expensive than the corresponding made by standard plastic matherial, it is also true that in the most of the cases, after carefull washing with milliQ water and ethanol20%  can be re-used many times. 

 

ProteoCool pills#24: Rapid fluorimetric DNA plasmid quantification on 96 welll plate

One of the most common methods for nucleic acid detection and quantification is the measurement of solution absorbance at 260 nm (A260) due to the fact that nucleic acids have an absorption maximum at this UV wavelength

When DNA is present in the sample a fraction of the ultraviolet light will pass through and an other fraction will be absorbed and the amount of the light absorbed is directly proportional to the nucleic acid concentration in the sample. Using the Beer-Lambert Law it is possible to relate the amount of light absorbed to the concentration of the absorbing molecule.

At a wavelength of 260nm, the average extinction coefficient is:

-        0.020 (μg/ml)−1cm−1, double-stranded DNA;

-        0.027 (μg/ml)−1 cm−1, for single-stranded DNA 

Spectrophotometric quantification is precise and with the advent of microvolume spectrophotometer (e.g  nanodrop) those allow to perform measurement using very small sample volumes (1-2ul) with-out the use of any sample support (e.g quartz cuvette)  it become the method of first choice for DNA plasmid quantification in the molecular biology laboratories.

DNA quantification with microvolume spectrophotometer is precise and allow to evaluate DNA purity and RNA but it is time consuming  (20-30’’ for sample) and therefore not applicable to the measurement of a huge number of samples in parallel.

Modern HT (High throughput) cloning platforms produce hundreds of DNA samples (plasmidic ans/or genomic) in parallel and using a multiwell based approach is certainly preferable to speed up the process. 

Since multiwell determination require at least 50-100 ul of  sample/well,  a preliminary sample dilution step is required to do not use the entire DNA sample for this step, but this may represent a problem, since the method sensitivity is limited.

For example: 

- If we consider that A(260)=0.1 using a spectrophotometer with 1 cm of optical path-lenght correspond to a dna sample with concentration of 5ng/ul. 

Generally the path-length in a multiwell plates is lower than 1cm 

For example 100ul of an half area UVclear 96 well plate result on a path length of about 0,67cm  (A(260)=0,1 with a 7,5ng/ul sample)

Therefore if we dilute 10ul of our MINI PREP to 100ul final and we read the ABS280 on a multiplate reader we will obtain a  detectable ABS (>0,1) only for samples with concentration >75ng/ul that is too high since in my experience the range of plasmid concentrations that are generally obtained with a 96 well plate mini kit is in the range 20-100ng/ul. 

In this post I would like to show you as using a common Fluorimetric stains  (in my case Lonza Gel Star) developed to bind DNA staining in agarose gel a rapid DNA quantification could be performed in 96 well plate format. 

Example:

DNA plasmidic quantification using Gel Star probe (Lonza)

2ul DNA sample in 100ul Gelstar stain diluted 10000 times (1X final concentration) in H2O 

plates: 96 well flat black (Greiner)

Instrument: multiplate reader (Tecan M200)   Ex:490nm; Em:530mn (gain:80)

A standard curve was built using an available commercial plasmid pRSET/BFP (Invitrogen) and serial dilution were performed to obtain a final DNA concentration range (2,5- 0,0025ug/ml) 

The fluorimetric methods using GelStar show linearity in a concentration range 0,01ng/ul to 0,625ng/ul.

Therefore the methods, using 2ul of dna sample (dilute in 100 of probe) could be direclty applied to the quantification of DNA samples in concentration range 0,5-30 ng/ul that is in the range of the sample that normally are obtained for 96-well mini kit dna preparation kit.

In case that DNA samples are more concentrated we can simple reduce the DNA volumes  (to 1ul or 0,5ul) used for the test.

Considering that fluorescence of the probe can depend from DNA size and origin (single or double strand) is it ever suggested to perform a calibration line with a standard DNA sample with similar size and origin respect the ones that we would like to quantify.

Of course differently to 260/280 nm quantification this methods do not allow to you to estimate sample purifity )in terms of protiens) or buffer contamination but i'm my opinion modern mini kits are quite reilable and in 99% of cases the sample quality is ok for the downstream applications (eg sequencing, E.coli trasformation)

In my experience, this method is very usefull to rapid quantification of high number of purified plasmid to use for sequencing, trasformation and protein expression. 

I have done those trials with GelStar probe since it was the one avaialble in my lab at the time of this test but i suppose that similar results can be obtained also with other simiilar probes (eg, Gelred, midori green, Sybr safe it the right Ex/Em wavelenght were selected. Since each probe is chatacterized from a different fluoresence quantum yielad is possible that a different probe may affect a little the limit of sensitivity. 

Please, DO NOT USE Ethidium Bromide!!

Fortunatelly today,  less toxic probes (as i already mentioned in the ProteoCool Pills n°4) with similar sentitivity and low cost are avaialble. 

ProteoCool Pills#22: Tips for cleaning the OMNISEC RALS/LALS flow cell if get dirty after the passage of biological samples

 

                                                                           OMNISEC 

is an advanced GPC/SEC system combining a pump, degasser, autosampler for mobile phase delivery and sample injection module with an integrated multi detector incorporating refractive index, UV/Vis, light scattering and viscosity detectors.

I have used the OMNISEC system to analyze the aggregation state of several biological samples (eg. recombinant antibody/protein preparations) in standard Phospate, MES or Tris buffers (pH range 5.5-8).

I found amazing the performances of the OMNISEC RESOLVE module, that thanks to the presence of a temperature controlled Autosampler (4^oC – 60^oC), allow to load in reproducible way an high number of samples and guarantee a very good throughput.

One of the main drawbacks (which i'm not sure if is it is common or not to other light scattering systems) that i found is the high tendency of the LALS detector to get dirty after the passage of some biological samples.

In this post i would like to share with you my experience about the cleaning procedure to use when you  see a strong increase of the LALS baseline signal after the passage of biological samples.

For example in the following video yoo can see the baseline LALS signal that was detected some months ago after the run of about 20 mab samples in a SEC coloumn (all the mabs were expressed from ExpiCHO cells, purified with MAbselect resin and buffer exchanged in PBS by desalting) :

If the RALS signal is not much higher than the optimal (aobut 80mV), the LALS signal was very high (optimal range is 200-300mV) and a cleaning procedure was required.

Since we do not observe any improvement (data not shown) from the passage of any routine cleaning solutions (methanol 10%, acetonitrile 10%, sodium azide 0,02%) suggested on pag106 OMNISEC SYSYEM Basic Guide manual provided with the instrument, we then decided to proceed with Deep cleaning (pag.105) using a 5% HNO3 solution:

        BUT UNFORTUNATELLY WE OBSERVE ONLY A WEAK REDUCTION IN THE LALS BASELINES SIGNAL

we than tested SDS (1% solution) which may be able to resuspend and remove protein aggregates/precipitate:

BUT UNFORTUNATELLY  ALSO IN THIS CASE WE DO NOT OBSERVE A REDUCTION IN THE LALS BASELINES SIGNAL

Finally we tested NAOH 0,1M solution, which is routinelly used for the cleaning of  several chromatographic resins  used for biologic purification  (e,g proteinA, proteinG, sepharose)

NAOH 0,1M works very well 

and restore the LALS BASELINES SIGNAL in the optimal range

 Thanks a lot to 

 Mirco Toccafondi 

ProteoCool Pills#21: Chromas a free tool to check your Sanger sequencing results

In the last 20 years several different software packages able to support the scientist in their molecular cloning activities  (e.g primer desing, sequence assembly, transaltiion, vector desing  desing to manipulate DNA and protein sequences  as CloneManager, SnapGene, VectorNTI (discontinued by Thermo from 2019)

Those software are fully integrated set of tools for e cloning simulation, graphic map drawing, primer design and analysis. They are charaterized by a huge number of functions and they require the payment of a licence. 

Are those advanced softwares striclty necessary for a molecular biologist that would like just to check the result of a gene cloning into a mammalian or bacterial expression vector ?

In my opinion NO! 

Those softwares could be certanilly useful (for example to map the primer annealing region, to desing the vector map) but not essential expecially if you have to produce a limited number of clones in parallel.

For example, in this post i would like to present youm  Chromas, which  is a free simple, easy-to-use sequence viewer and editor (able to open chromatogram files(.ab1) produced from automated Sanger sequencers) that could be used to check your sequencing results.

7A. Allign the Chormas sequence with the “Theoretical” DNA template 

using ClustalW or ClustalOmega

or 

7B. Translate the sequence in AA format 

 (Generally I’m using the Translate Tool available at the Expasy web server) 

and allign the translated sequence with the “theoretical AA” template

 using ClulstaW or ClustalOmega

If the sequence show some point mutation or shift, do not  discard the clone but check carefully the chromatogram to see if the mutation/shift is real or is it due to a non-correct Chromas assignment that may happen in case of:

-  overlapping peaks:

- peak with low intensity;

- a sequence regions with multiple residues of the same species;

  

Other similar softwares:

Finch TV

ProteoCool Pills#14: Hints for cell propagation and recombinant protein expression using Expi-CHO and Expi293 (WT and GNTI-)

For many years the application of the traditional adherent mammalian cell cultures for recombinant protein production was difficult and applicable only in those approaches requiring very low protein amount (ug’s) because cell scale up was very difficult, transfection not very efficient and the final volumetric yields (mg/litre) very low. 

This limitation forces the scientist to use other simple eukaryotes expression systems as Pichia Pastoris or Baculo virus for the production of mammalian protein in mg scale.

However, in the last 20 years some technological improvements as:

 1) The adaptation of several cell lines to suspension cultures;

 2) The development of more efficient transfection agents;

 3) The development of serum free media where the cells are able to growth at high cell density;

Improve drastically the performances of the mammalian expression systems;

As I already mention in previous presentations, theExpi293 and Expi-CHO date, represent 2 of the most performing mammalian transient expression systems. 

If it is true that those systems are quite expensive, on the other hand they are very simple to use and guarantee, high yields also in shake flask. 

For some recombinant protein, Expi293 in shake flask were able to provide me up 300mg/yields. 

Values that until some years ago were reachable only using E. coli expression in high cell density set-up (e.g. feed-batch fermenters)

Expi-CHO are able to provide up to 100-200 mg/l of full length recombinant antibodies. Full length antibodies are big (150KDa), composed from 2 chains and contains several disulfide bonds, their expression in bacterial expression system is very challenging.

However, to reach those results details, as;

-             - The selection of the optimal shake flask format and right culture volume;

-              -  The selection of an antibiotic not affecting cell growth and protein expression;

-                The selection of the optimal signal peptide for protein secretion into the cell surnatant;

can make a huge difference!!

The selection of the right shake flask and culture volume

Expi293F and Expi293 GNT- cells :

In small scale (up to 60ml culture) Expi293 are not so sensitive to the flask format and presence of baffle:

For example, in our experience Expi293 are growing well in both

-        Erlenmeyer flaskscup vented plain (cost ~ 8€/flask for 125ml format)

-        Erlenmeyer flasks cup vented baffled

-        Thomson Optimun growth flaks  (cost ~ 8€/flask for 125ml format and 30€/flask for 1,6l)

Optimal culture volumes: 

125ml flask volume for 30ml Expi293 transfection and 250ml flask volume for 60ml Expi293 transfection

For culture scale up (eg 200-500ml culture),  the Thomson Optimun growth flaks eems to perform better probably  because it guarantee better CO2 and oxygen exchange.

My preferred format is 200-300ml transfection in 1.6liter flask and generally we do not exceed the 500ml single transfection (using a 2.4 litre flask) because in high volumes we more frequent bacterial contamination was observed, therefore in case of large culture volume e.g. 1-2liter, I prefer to split the culture using more flask in parallel.

ExpiCHO:

ExpiCHO show an high tendency to aggregate and are much more susceptible to  the flask format.

Thomson Optimun growth flaks https://htslabs.com/og/ that contain small baffles seem to represent the best compromise and are the format more able to prevent the cell aggregation during the cell maintenance passages.

N.B: Expi-CHO are able to growth up to very high cell density (>10milion/ml) but to avoid cell aggregation is it better do not exceed the 6 milion/ml during the cell propagation passages

Optimal culture volumes: 

   125ml flask for 25ml transfection 

   250ml flask for 50 ml trasfection

    1,8liter flask for 150-300ml tranfection

In all cases we use the max titer protocol à 8day of trasfection at 32°C

The selection of the right antibiotics to prevent cell contamination.

The Expi293 and ExpiCHO Thermofisher manuals do not suggest the addition of any antibiotics to prevent bacterial contaminations. However after that we experienced some culture contaminations, considering the high  cost of the reagents (more than 1000euro/Liter)  we decided to perform some  transfection trials in the presence of  antibiotics to see if they affect or not the cell viability, growth rate and recombinant protein expression

Expi293 are not affected by:

-        Pen/strep (thermo cod. 15070-063) diluted 1:100 (50 unit/ml penicillin and 50ug/ml Streptomycin)  

-        Gentamicin (Sigma cod. G1272) diluted 1:200 (50ug/ml)

Generally, we use for cell propagation and transfection the Pen/Strep 

Expi293 GNT- are not affected by Gentamicin (Sigma cod. G1272) diluted 1:200 (50ug/ml) while reduction in cell growth rate and viability was observed in presence of Pen/Strep;

Expi-CHO are not affected by Pen/strep (thermo cod. 15070-063) diluted 1:100 while reduction in cell growth rate and viability was observed in presence of Gentamicin diluted 200 times.

 

 

 

 

 

ProteoCool Pills#13: Densitometric Protein quantification from SDS-page using the Image J free software package

 Several different methods are currently available to perform quantification of purified recombinant proteins and antibodies

There is not a best, universal method, the provide a reliable result for all the proteins;  

Each methods have it some prons and cons and its applicability depend from the intrinsic properties of the target protein.

For example:

UV quantification that exploits the properties of aromatic amino acids (tryptophan and tyrosines) to absorb energy around 280 nm is fast and require limited amount of sample but cannot be performed with proteins that do not contain aromatic amino acids or with buffers with an intrinsic absorbance in the UV regions.

Colorimetric-fluorimetric assays as Bradford, BCA, Nanoorange are susceptible to buffer compositions (eg BCA is not compatible with reducing agents) and to extrapolate quantitative results, the comparison with a  calibration line is required Results may change a lot on the basis of the protein that is used to build the calibration line (generally BSA) because different proteins may show different response in function of their aminoacidic composition or stability of their conformation in presence of the dye.

gg: Bradford assay is less sensitive to full length antibodies (igG) than BSA (see fig 2 page 6 ) and therefore in case you would like to use Bradford assay to quantify a monoclonal antibody (mab) a calibration with a commercial mab is required.

In some unlucky cases, for those proteins that do not contain hydrophobic amino acid and shows low response to colorimetric assay (due to strong conformational stability of presence of post translational modifications, eg hyper glycosylation) all the previous methods may not be reliable and densitometric analysis from SDS-page may represent a simple and cheap alternative.

Quantitative densitometry of proteins from SDS-page stained with colorimetric reagents (eg  coomassie blue) require a software to perform image processing,  extrapolate peak area and correlate it with the sample concentration.

To date most of the commercial gel documentation systems are supplied with their Image analysis Software able to perform band intensity determination.

However, if those Gel acquisition systems are still essential for acquisition of agarose gel images, high quality images of SDS-page gels stained with Coomassie can be obtained using modern smartphone those carrying high resolution camera.

ImageJ (NIH), a public domain program from the National Institutes of Health downloadable at https://imagej.nih.gov/ij/download.html can be used to analyse the SDS-page images.

1. Open the gel image

On the gel selected for this example, we load several dilutions of a purified protein sample with unknown concentration (to be determined) and several know amount of BSA required to build a reference calibration curve

2. Select rectangle in the AREA SELECTION TOOL

3. Choose the 1^st  line, select the rectangle tool, and draw a box around the lane, 

making sure to include some of the empty gel between lanes and white space outside of the band. 

When creating the selection, drag with the shift key down to constrain it to a square.

4. Define the 1^st  line: Go to Analyze→Gels→Select first lane

      5. Select the 2^nd line

        Make sure your cursor shows as an arrow, grab the rectangle you just made, and drag it to the next lan

DO NOT DRAW NEW RECTANGLES! You must drag the same rectangle you just made because to compare the band you have to use the exact same size originally defined area in Lane 1.

6. Define the 2^nd line: Go to Analyze→Gels→Select next lane

 

7.  Repeat the step 5 and 6 since all the line (sample and standard dilutions) are selected and numbered

 8. Go to Analyze→Gels→Plot lanes 

 

A new windows containing an histograms for each line will appear

9. Drag two fingers on the mousepad to scroll up and down and navigate the grids

The peaks in each grid correspond to the intensity of the bands in the lane

 10.   On the ImageJ interface, select the "line" button (red arrow) to define the peak baseline

     11. Draw a line at the bottom of the peak that represents the baseline of your peak and it allow to define the area of the curve.

12. Drag two fingers on the mousepad to scroll down and keep drawing all the single lines to define the curves in your standard and protein of interest lanes.

 

  13. Once you draw a baseline for each peak, on the ImageJ interface, select the "magic wand" button (red arrow)

 

 14. Click on the line defining the area of the curve of the first peak

 A "Results" window containing the measured area will appear

  15. Drag two fingers on the mousepad to scroll down and define the area of all peaks with the defined baseline

16. In the Result window Go to File→Save as

ans Save your Results in .csv format so that you can transfer the measurements to excel to generate the standard curve (linear regression analysis) and determine the concentration of your protein sample.

A Possible mistakes:

When you draw the peak baselines (point 10-12), the line has to interpolate both “feet” of each peak 

to correctly define and measure the peak area. 

The same analysis could be used to determine band intensity and extrapolate dna or rna quantification from agarose gel. 

However in my opinion the limited linear range of densitometric analysis and low reproducibility in gel load and coloration make this quantification approach not very precise and have to be applied only when better alternatives (as 280nm quantification for DNA) are not available.