I'm Manuele Martinelli, a chemist with almost 10 years of experience as a senior R&D scientist (focused on protein production and characterization). During my time in the lab I've had the opportunity to test a lot of instruments, procedures and reagents, and I'd like to share some Cool Protein ProtoCols (ProteCool!)with you in the hope that they help you with your experiments.Since I was born strabic, I always had a different point of view, which I want to share with you! :-)
Chain stores with furniture, DIY, decoupage hobbies as
IKEA, OBI, Leroy Merlin, Bricoman
can be a great source for funny and cheap items useful for improve the laboratory organization
Here i report just some examples:
1) SANDVIVIA OVEN MIT
made in silicone which provides a firm grip and is heat-insulating.
It allow to manipulate and move the hot agarose gel and/or hot culture media removed from microwaves or autoclave.
Avaialble from IKEA (Italy: 3euro - US: 4 dollars )
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2) VEKSEN CART
It fits in the smallest of laboratory, but there’s plenty of space on the shelves for all for all those staff that you need to have on hand when you work on the laminar hood
Assemble the trolley quickly and easily by clicking the parts together without any tools.
Available from IKEA (Italy: 10 euro; US: 9 dollars)
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TIKSEN Hook with suction cup.
Alternative clothes hanger solution for lab coats
Available from IKEA (Italy: 7euro/4p; US: 5dollars/2p)
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BEVARA Sealing clip.
Cheap clips for use with Dialysis Tubing
Available from IKEA (Italy 1,5euro/10p: US: 2 dollars/10p )
DSF
(Differential scanning fluorimetry previously named also Thermofluor)
is a very simple tecniques that exploit the ability of a fluorescente probe, typically either SYPRO Orange or 1-anilino-8-naphthalenesulfonate (ANS) – that is quenched in an aqueous environment but becomes strongly fluorescent when bound to exposed hydrophobic groups of a protein. Therefore, the thermal unfolding of a protein of interest in the presence of such a dye, can be followed spectrophotometrically.
DSF is a 96 well based assay that could be performed on a RT-PCR thermocycler, it is inexpensive, fast, and require relatively little sample. All these advantages have made this approach attractive for screening applications in drug discovery and also for protein stability formulation
In the ProteoCool n°24 in suggest as in my opinion the DSF may become an essential assay todetermine lot to lot concistenciy in QC department of company producing recombinant proteins (e.g recombinant vaccine and/or enzymes)
In this post i would like to share with you 2 examples that show as DSF may play an important role also in antibody screening, optimization and developability assesment.
DSF thanks to its excellent throughput and minimal material consumption (about 50ul of antibody at 0,3 mg/ml for each point) allow to readily compare the thermal stability of:
- different antibodies in the same buffer:
- different Fab mutants or Fc mutants in the same buffer;
- different antibody formulations; (optimize pH, salt concentration, excipients)
Example 1:
Comparision of thermal stability of different mabs
Full length human IgG1-Clk mabs in PBS may show 1 or 2 separate transitions on the basis of the Fab stability.
Mabs with lower Fab stability show in general a single transition while mabs with high stability show 2 separate transitions, 1 at about 69°C for the Fc and 1 at higher temperature for the Fab.
In recent years the enhancement or elimination of the Fc-effector function has led to a growing interest in Fc-engineering, to give antibodies specific mechanisms of action and therapeutic properties.
Even if several Fc modification, as:
- LALANA, LALAPG, LALAGR mutations to remove effector function;
- SDIE, GAALIE mutations as well as afucosilation to enanche the effector function
were already reported, few data about developability of those solutions are still available and there is a lot work to do to select an enanched Fc combining the best effector function and good manufacturability, stability and pharmacokinetics.
Those are just 2 examples that show as in short time and using a limited amount od samples, thanks to DSF we can obtain essential informations to guide our decision in antibody during the screening, desing, formulation phases.
Of course, thermal stability itself do not Guarantee that the selected antibody has good developability profile but some of the following other tecniques as:
- DLS;
- Self-interaction determined by BLI;
those provide informations about antibody aggregation propensity
- Hydrofobic interaction chromatografy (HIC)
those provide informations about antibody hydrofobicity
have to be peformed to complement the DSF data.
A great thanks to
Luca Sorrentino
In the Video Section of ProteoCool are actually available more than 30 different video tutorials about molecular biology, protein expression and characterization.
All those video can be easily downloaded using the Video DownloadHelper tool available on Mozilla Firefox browser. No cost or restriction are associated to the ProteoCool video use or sharing.
I just ask you to cite, ProteoCool, in case you will share or use part of it for build new material.
In this post I would like to show you, how video download from a Blog (not only PRoteoCool but all the blogger.com blogs is possible using the Mozilla browser
How yu can do it:
- Open Mozilla Firefox browser;
- Check if the Video DownloadHelper is already installed in your browser
The presence of the Video DownloadHelper is identified by the presence of an Icon that shows 3 balloons (in gray scale) located on the Firefox toolbar.
If Video DownloadHelper is already installed, you can choose your video and download it in few minutes following few steps:
1. Access to the blog from which you would like to download the video (e.g. PRoteoCool)
- Click on the VideoDownload helper icon and proceed with the installation by
following the different instructions.
- Once the 3 balloon icon will appear on the top of your browser, Downloading videos
from the Blogger site is now possible.
Go to the step 1
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:
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.
Static light scattering (SLS) is a technique to measure absolute molecular weight using the relationship between the intensity of light scattered by a molecule and its molecular weight and size.
Some SLS technologies exist: multiangle light scattering (MALS), right-angle light scattering (RALS), low-angle light scattering (LALS) and RALS/LALS hybrid systems
MALS or RALS/LALS when coupled with other detectors (eg UV-vis, RI, densitometry, fluorimeter) in an advanced GPC-SEC system can be applied to investigate solution properties, stability testing and process development for different kind of conjugated and unconjugateed biologics
Light scattering detectors (low-angle LALS, right-angle RALS, or multi-angle MALS) are very sensitive to the presence of particulates, when those are used for molecular weight detection in chromatography. Unfortuantelly even the best columns can shed some particulates from their packing material. Although undetected by most conventional detectors, such as UV and RI, these particles scatter significant amounts of light, produce noise that affect the light scattering signals and baselines. To mitigate this, the MALS, LALS/RALS sistems include an in-line coloumn filter that can significantly improve the quality and thus the accuracy of both the data and results.
In this post i would like to share with you some data acquired loading different mabs in a Cytiva Supedex200 increase 10/30 coloumn in the OMNISEC instrument equiped wih the LALS/RALS detector in the presence/absence of post coloumn filter.
First of all we compared the baseline signals that can be obtained
- Without filter;
- With a 0,2um nylon filter;
- With 0,2um cellulose filter;
The baseline signals suggest that nylon filter is the best in terms of signal/noise ratio
Is it really the best choiche for analisys of protein/antibody preparations?
To assess it, a 2% BSA standard and 2 different purified monoclonal antibodies (igG1 human) were loaded in the superdex200 10/30 increase coloumn and analized with the OMNISEC instrumentation
In all the 3 cases, the Nylon filter guarantee the best signal/noise ratio at LALS but it seems to mask the presence of a significant fraction of high molecular weight proteins.
This was most evident for the mabs, since the dimeric-trimeric and MW aggregates were not detected, even if at UV and RI, using the nylon filter.
ThIs differences may lead to overtimating the quality of a certain mab or protein preparation and it can lead to false positive results in case of stability studies that would like investigate the mab/protein aggregation propensity under stress (as acid pH, 37°C, freeze/thaw)
For this kind of studies cellulose filter seem to be the best compromise between quality of LALS signal and recovery of the protein polimers.
As a general comment: Often the diffence is in the details! It is important be able to critically review the Positive results to distinguish the real positives than false positives!
Suggested links:
https://www.materials-talks.com/how-to-change-the-light-scattering-post-column-filter-membrane/
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 (4oC
– 60oC), 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)
Thanks a lot to
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 
