Bacterial Cell Culture - Science method

Different labs use different wavelengths to measure, usually from 590-650. They all work fine so it doesn't really matter so long as you consistently use the same. Traditionally 600nm has been used but not by everyone, I think because it matches the old color filters of Klett meters that used to be used.

When you get to optical densities above 1, the amount of light being scattered vastly exceeds the amount passing through, so the accuracy of the measurement goes way down. As the other responders said, the solution is to dilute your culture and take your reading so that it is below 1.

Hi... Were you able to coat cells? If yes, then please share the protocol.

Swapnil Srivastava then, you could have carbohydrates in your samples that do not form furfural in the dehydration step, so the response is negative for them.

Cosmetics and foods that canoit be reliably extrudedd/plated by the spiral plater.

The age of a bacterial culture can greatly affect DNA isolation. Younger cultures, which are typically in the exponential growth phase, tend to yield better results because the cells are actively dividing and less likely to have degraded DNA. In contrast, older cultures may have more dead cells and debris, leading to lower quality DNA and potential contamination. Additionally, cells in older cultures might be harder to lyse due to changes in cell wall structure, and the presence of extracellular nucleases can degrade the DNA further. Therefore, it's usually best to use younger bacterial cultures for DNA isolation to ensure you get the highest quality and quantity of DNA.

According to Pikovskaya R, Pikovskaya RI. Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Microbiol. 1948;17:362–70,

PKO liquid medium consist of NaCl 0.3 g, Glucose 10 g, KCl 0.3 g, Ca3(PO4)2 - 5 g, (NH4)2SO4 0.5 g, MgSO4· 7H2O 0.3 g, MnSO4 0.03 g, FeSO4· 7H2O 0.03 g, distilled water 1000 mL, pH 7.0–7.2, agar 15-20 g, autoclave (30 min).

Indeed, 50% glycerol slants serve as effective storage solutions for bacterial cultures at -20°C, leveraging glycerol's cryoprotectant properties to safeguard cells from ice crystal formation during freezing and thawing. To prepare them, mix glycerol and sterile water equally to create a homogeneous 50% glycerol solution. Inoculate the slants with the desired bacterial culture and let them grow before adding the glycerol solution to cover the growth. Seal the slants and store them at -20°C to maintain viability over months to years. To revive cultures, streak cells from the slants onto agar plates and incubate under suitable conditions. Although glycerol enhances viability during storage, it's vital to handle cultures carefully and adhere to proper storage and revival protocols to ensure their viability.

I add 50mg/L of Nystatin as a methanol solution (or rather suspension). The 50mg does not dissolve completely in 1mL of methanol but the suspension is homogenic so if you add this to warm agar (with stirring) then you get a somewhat even dispersal. Hope that helps...

If in doubt make serial dilutions and measure those. If you chill your culture in a fridge the OD should not change much in the time you take to measure the OD.

So far, the second derivative of the bacterial growth curve was more informative than average rate of growth.

Hello, it can happen, the absorption at 600nm of your media can drop during the time if you have some non visible particles,which can precipitate during storage in thermostats it can cause drop of absorption. You can perform filtration of media before sterilisation or after with the use sterile sterile conditions to avid it. Or it can be caused by color change of media during the storage.

There is no exact trash hold for absorption at 600 nm it variate depends from type of nutrient media. But if you have microbial grow it tube 10*5 or above this drop can be negligible.

Good luck!

My take is that starter culture preserved at 4oC for up to 24 hours will not have any significant population change. This is due to inactivation of microbial enzymes. However, if left for longer than this period, cell start entering a decline phase and therefore a lower population in the starter culture.

Agar well diffusion method or disk diffusion method can be used check phytochemicals against disease causing bacteria

I agree with Phil Geis that Petri dishes is not the best way to store your strains. Frozen cultures at -80 is great for long term storage in glycerol, although if you are constantly using these strains many labs have traditionally kept agar slants, one per strain, which are usually viable for a month or a few months. But you should always have a permanent backup.

Dec.9, 2023

Dear Xieyue,

I don't know what polysaccharides besides pectin you are working with, but most polysaccharides are pretty stable and can be autoclaved. Check that the pH is near neutral, as polysaccharides will be hydrolyzed by the heat of autoclaving at low pH. If you are concerned that the polysaccharides will form gels at high concentrations, you can use lower concentrations, e.g., 5-10%, and autoclave them in the liquid medium you are using. This way, when you add the polysaccharide to the liquid medium, you won't get a dilution effect.

I hope this information helps you.

Bill Colonna

Dept. Food Science & Human Nutrition, Iowa State University,

Ames, IA.

There are several possible reasons why no emulsion formation may occur in a beta carotene/linoleic acid bleaching assay. Some potential reasons include:

1. Inadequate mixing: If the beta carotene and linoleic acid are not mixed thoroughly, emulsion formation may not occur. Proper mixing is essential for the formation of an emulsion.

2. Incorrect ratio of beta carotene to linoleic acid: The ratio of beta carotene to linoleic acid may not be optimal for emulsion formation. Adjusting the ratio of these components may be necessary to promote emulsion formation.

3. Insufficient energy input: Emulsification often requires the input of energy, such as through stirring or homogenization. If insufficient energy is applied, emulsion formation may not occur.

4. Incompatible solvents: The solvents used for dissolving beta carotene and linoleic acid may be incompatible, leading to a lack of emulsion formation. Using compatible solvents or adjusting the solvent system may be necessary.

5. Contaminants or impurities: Contaminants or impurities in the beta carotene or linoleic acid samples could interfere with emulsion formation. Purifying the samples or using higher quality reagents may be necessary.

6. Temperature and pH conditions: The temperature and pH conditions of the assay may not be suitable for promoting emulsion formation. Adjusting these parameters could help facilitate emulsification.

7. Other factors: Other factors such as the presence of surfactants, stabilizers, or inhibitors could also affect emulsion formation in the assay.

It is important to carefully troubleshoot and optimize experimental conditions to promote emulsion formation in a beta carotene/linoleic acid bleaching assay.

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ChatGPT DALL-E Bot, [11/13/2023 6:58 PM]

Selecting the solvent for extracting unknown compounds using a Soxhlet extractor can be a trial-and-error process. It is important to consider the polarity of the compounds you are trying to extract and choose a solvent that will effectively dissolve them. Additionally, the boiling point of the solvent should be lower than that of the compound being extracted in order to facilitate efficient extraction.

One way to determine the appropriate solvent is to perform a preliminary thin-layer chromatography (TLC) analysis of the unknown compound. TLC can help identify the polarity of the compound and provide insight into which solvent may be most effective for extraction. By comparing the results of TLC with different solvents, you can determine which one yields the best separation and select it for use in the Soxhlet extractor.

It is important to note that while TLC can provide valuable information about solvent selection, it may not always accurately predict how well a particular solvent will perform in a Soxhlet extraction. Therefore, it may be necessary to conduct further experimentation with different solvents in order to find the most suitable one for extracting unknown compounds using a Soxhlet extractor.

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ChatGPT DALL-E Bot, [11/13/2023 6:59 PM]

To perform sunglint correction and water column correction on WorldView-3 imagery in ArcGIS Pro, you can use the following steps:

1. Sunglint Correction:

- To correct for sunglint, which is the reflection of sunlight off the water's surface, you can use the Atmospheric Correction tool in ArcGIS Pro.

- First, open the WorldView-3 imagery in ArcGIS Pro and navigate to the Image Analysis tab.

- Click on the Atmospheric Correction tool to open it.

- In the Atmospheric Correction tool, select the WorldView-3 imagery as the input and choose the appropriate parameters for sunglint correction, such as selecting "Sunglint" as a correction option.

- Run the tool to apply sunglint correction to the imagery.

2. Water Column Correction:

- To correct for water column effects, which can cause distortion in underwater features, you can use remote sensing techniques such as band ratio or spectral indices to account for water column effects.

- In ArcGIS Pro, you can use tools such as Band Arithmetic or Raster Calculator to create band ratios or spectral indices that account for water column effects.

- Once you have created the appropriate band ratios or spectral indices, you can apply them to the WorldView-3 imagery to perform water column correction.

By following these steps, you can perform sunglint correction and water column correction on WorldView-3 imagery in ArcGIS Pro to improve its accuracy and usability for various applications such as environmental monitoring, coastal management, and marine research.

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ChatGPT DALL-E Bot, [11/13/2023 7:03 PM]

Yes, topographic correction is needed for Landsat 8 level 2 products in calculating NDVI for mountain areas. Topographic correction helps to account for the effects of terrain on the reflectance values captured by the satellite sensor, particularly in areas with varying slopes and elevations. This correction is important for accurately calculating NDVI in mountainous regions, as it helps to minimize the influence of topographic effects on the vegetation index values.

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ChatGPT DALL-E Bot, [11/13/2023 7:07 PM]

To extract a sample for LC-MS (liquid chromatography-mass spectrometry) analysis, follow these general steps:

1. Sample Preparation: Begin by preparing your sample according to the specific requirements of your analysis. This may involve homogenizing or diluting the sample, depending on its nature.

2. Extraction: Choose an appropriate extraction method based on the properties of your sample. Common extraction methods include liquid-liquid extraction, solid-phase extraction, and protein precipitation.

3. Centrifugation: If necessary, centrifuge the extracted sample to separate the supernatant from any solid particles or debris.

4. Filtration: Use a syringe filter or centrifugal filter to remove any particulates or impurities from the extracted sample.

5. Transfer: Transfer the filtered sample into a vial suitable for LC-MS analysis.

6. Storage: Store the prepared sample in appropriate conditions (e.g., refrigerated or frozen) until it is ready for analysis.

It's important to note that these steps are general guidelines and may need to be adjusted based on the specific requirements of your LC-MS analysis and the nature of your sample. Always refer to the standard operating procedures and guidelines provided by your laboratory or analytical instrument manufacturer for best results.

Make sure that the water you use is distilled or miliQ, no tap water should be used for washing or diluting the glycerol. Also, chill the cuvettes in ice before using them.

Are you sure that the parameters you are using for electroporation is correct? Pay attention to the gap size of your cuvette and modify electroporation parameters accordingly.

Check this info, might be helpful: https://www.btxonline.com/media/wysiwyg/protocol_db/Electroporation_Optimization_Guide.pdf

Have you considered doing gentle (-partial-incomplete) digestions with lysozyme ? or with other kind of "Enzymatic dispersion of biofilms".

Best wishes

I guess your doubt is related to Intra-Cellular metabolites (I guess you have chosen secondary metabolite compounds instead). You can have a look at my recent publication, which was published in RSC Molecular Omics recently. It may drive you to some extent in your research focus.

Well could be the non-structural protein has a temperature dependent interaction with something in your expression vector. NS1 is known to affect lipids while NS4 Modifies the ER.

Thank you, Péter!

There is a similar question.

You can use the TRIzol method which is a cost-effective method and I found it way better than using a commercial kit. If you are using gram-positive bacteria, add the freeze-thaw step (freeze at -80 degrees for an hour after adding TRIzol and then thaw) to degrade the cell wall of the bacteria and to get a better yield of RNA.

I know I've used 125 mL baffled shake flasks (Erlenmeyer), which is definitely a good option if you're looking to reuse them. The supplier Chemglass appears to offer baffled flasks down to 50mL. I would just verify it's an appropriate type of glass for your purposes and cleaning methods. If you really like the cell culture flasks, I'm fairly sure those can also be made with glass, which could be autoclaved, but reusing them is apparently a bit contentious depending on your application (Look up "Can tissue culture flasks be reused?" by Alexis Weston on ResearchGate; one of the popular answers contains some helpful resources). However, it should be fine in the case of bacteria. I'm not sure if the vented caps can be autoclave-safe or not, so it would be good to check with the supplier for appropriate cleaning methods.

When you are ready to culture the stored samples, thaw them by transferring the cryovials from the -80°C freezer to a 37°C water bath or under room temperature conditions. Use appropriate sterility precautions during thawing and subsequent culturing processes.

It is important to note that different bacterial species may have different sensitivity to cryopreservation and may require specific modifications or optimizations in the cryopreservation protocol.

Thanks again Fatemeh Gharahdaghigharahtappeh mam for taking time out of your schedule to help me out.

Try increasing the salt concentration. Higher salinity can make some motile bacterial species become sessile.

It was probably degrading during the crushing of the leaves or if the sample was frozen in storage. On cell lysis dna degrading enzymes are released and there is some cutting of dna until the enzymes are degraded. When you run the gel it just shows you the amount of degradation as a smear. This is not usually a problem if you just want to do pcr when degraded dna amplifies perfectly well. If you are sure that it is the electrophoresis that is causing the degradation then this can happen when the buffer in the gel tank is re used many times and gets warm and bugs grow in the buffer and release enzymes that get into the gel and can degrade dna and rna

What strain is this supposed to be?

Hello Neha Ijaz

Yes, if you mix bacteria (in PBS) in broth, it should grow provided you have not left the bacteria in PBS for long. As you already mentioned that bacteria cannot grow in PBS because it is not a bacterial medium. PBS simply provides a conducive environment for bacterial cells to remain stable only for a short period until they are transferred to a more suitable environment (liquid bacterial media) for their growth.

Best.

FAU and NTU are unit used to measure scattering by suspended materials. The link to absorption is complicated. For example, if you had only absorbing dissolved material in your sample (e.g. tea) you will measure 0NTU. The number of NTU will go up if you added a white scatterer (e.g. plastic beads) particularly for non-absorbing beads. I hope the above makes sense to you. If the measurement setup is one of attenuation - both scattering and absorption cause attenuation, issues such as wavelength of measurement, acceptance angle of detector etc' could cause large variation between what you assume to be NTU reading and absorption.

Best, Emmanuel

This is just basic math. If your starting concentration is 100mM and you need a final concentration of 0.5mM, that would be a 200 fold dilution. So 1/200 x 500 = 2.5. Or the general equation would be 0.5 x 500 / 100 = 2.5

Hello, before injection of a small volume into HPLC just add 4N sodium acetate 1/10 volume and freeze the sample to precipitate the proteins, centrifuge the sample at 10000g, and then inject supernatant.

Robert Adolf Brinzer

Thanks dear Dr.Brinzer

Since culture media can be expensive, it is important to identify alternative options or reduce the amount of agar used in laboratories with limited resources. In this regard, Arnan-Parh et al. have investigated the use of cowpea as an affordable alternative culture medium. Additionally, some studies have utilized starch or vegetables as a cheaper growth medium for bacteria and fungi. This study seeks to replace the nutrient source with a protein formulation using locally available, cost-effective legume seeds such as green gram, black gram, cowpea, and soya meat. These seeds are rich in protein and can serve as a viable substitute for traditional nutrient sources in culture media preparation.

Reference: Arulanantham, R., Pathmanathan, S., Ravimannan, N., & Niranjan, K. (2012). Alternative culture media for bacterial growth using different formulation of protein sources. Scholars Research Library. https://www.scholarsresearchlibrary.com/articles/alternative-culture-media-for-bacterial-growth-using-different-formulation-ofprotein-sources.pdf

Dear Samridhi, if you do the streak plate method for pure culture formation then no any matter of contamination. After pure culture formation , you do the sub culturing of your sample only 2- 3 times, then preserve the culture at 4 degree celcius.

For growing in liquid culture, you add once. E. coli and other standard bacteria will reach exponential growth quickly so no need to add again.

Same goes for plates. You add once right before pouring the plates. If needed, you re-streak onto fresh plates to avoid satellite colonies.

Talk with a postdoc or other graduate student or your PI.

If you really need to wait a month then I would run the tests that William F. Hansen suggested, otherwise you really will not for certain about the accuracy of your data. However this is likely to be true if your interest is only in total count. If you are seeking to determine the presence or absence of specific microbes, they might be very different in their survival in storage in water. While some will undoubtedly survive for a month, I strongly suspect that many others won't.

It is somewhat arbitrary but convenient value. You want to be sure to not use too much of the primary culture for inoculation so that you don't carry over waste products and signaling molecules from the old culture. And you don't want to dilute it too far back otherwise you have to wait longer for the secondary culture to grow. We often used a 100-fold when we wanted to use the secondary culture fairly quickly and 1000-fold when we wanted a bit of a delay or to capture the cells early in exponential growth. It also makes the math easy!

Kristova Yubilius Indrataruna I have two suggestions for you: 1) Use ASNIII medium for marine CB: BG-11 was elaborated for freshwater CB; 2) Use very purified agarose (like Kim Sea) instead of either agar or gellan gum because very purified agarose does not support the proliferation of heterotrophs. Good luck. Igor Brown

Yes you can.

I think the issue is that when you have removed a significant volume of the experiment then the conditions may be changed by the volume changes. For example the level of aeration may change, mixing rates will differ etc. So if by the end of the experiment you have removed 50% or 70% of the volume, the samples collected at the later time points will have experienced a different environment.

There may be some conditions or experiments where that does not matter, but it could be a concern. I doubt the 10% is any sort of exact number, just a rule of thumb.

To partially answer my question (in case anyone will stumble upon this in the future), 2mM PC does not dissolve in aqeous buffer. A detergent like bile salts for example is required to bring it into solution

hi ,

Yes I took 500ul from Glycerol stock to FResh Medium (in conical flask ). I stored 10-15 flask containing 100ml medium as Reserve ! I use it whenever I need ..

I will try to remake agarplates and try nextweek ,Thanks

You can also use the intermittent heat treatment method for the elimination of spores.

Well the recipe is several pages long, so I am not sure if it makes sense to go over that:). My only concern is whether the SCFA supplement needs to be made or can be bought. If you make it out of salts, can you please suggest the protocol that you use for that?

As you told about the cell reduction after induction it may be caused by gene toxicity. In that case you can try PlyS BL21 E.coli variant which can tightly express toxic genes too. And one another thing you can do is change those parameters as temp , IPTG concentration to check the induction . Do it alternatively in small culture first then go for large culture. And one thing what's is your protein size and PI that also important in case of expression.

Check the expiration date on your kit (and if anyone noted when it was opened). Kits tend to expire in 1 year after opening. If anyone left the wash buffer open (or closed poorly) then the loss of ethanol will make the yield decrease (the ethanol helps keep the plasmid from eluting during the wash step)

Maybe try a smaller scale prep (like mini) just to make sure you are able to get a good yield from less involved plasmid extraction

Hi Leonard,

I'll preface by saying that I don't know much, but if it's at dead volume, then this compound could just be a mixture of small hydrophilic compounds. I've seen something similar when analysing synthetic peptides - hydrophilic amino acids elute before 3 minutes running a water- organic 60 minute gradient.

Also, I don't think I would consider 500kDa "small", nor a peptide, 500Da on the other hand..

Hope this helps!

Gordana Zavisic

Thank you very much for the observations!

Normally, I leave the Petri dishes with the culture in the fridge for a maximum of 3 to 5 days.

Previously, I already tried using oil as you advise me and it grows well. Even without using oil and filling the test tubes to the top with MRS medium it gives good growth.

Interestingly, the problem occurs when trying to subculture from the previous grow. I have not found information on whether it requires special handling conditions.

I haven't tried incubating the tubes without shaking as you mention, I'll try that option.

Thank you again!

My suggestion is also to use fresh sheep blood in the medium.

Diesel per se is a complex mixture. "% biodegradation" can be misleading as some fractions may be readily metabolized and some not.

Thanks Jorgaq, but I was looking for something cheaper than a kit.

How old is the media you're subculturing it to? Some people in our lab thought our Enterococcus faecalis freezer stocks were going bad but we eventually figured out that E. faecalis stops being able to grow in BHI once it's been left at room temperature for about a month. That stopped happening when we started putting prepared BHI in the refrigerator for long term storage. It might be worth remaking your media if you haven't already - lactic acid bacteria tend to be fussy about their nutrient requirements.

Iam interested of knowing about this project, as long you can share the information with me.

In other, most bacteria can be measure with microscope

Tap water also has pharmaceutical residues and metabolites. I don't know what is the status in your country but in Kuwait, we do have very reliable data that we have often at trace levels.

Tey one control without inoculum, so that you can find whether it is because of inoculum or agar,

I too faced this kind of issue I found some white color spots after, Instead of troubleshooting I opened a new agar jar and compared the presence and absence of the spots and processed with the new one,

Hi!!!!

Please do standard for determining protease activity with a substrate and compare your bacterial culture's protease activity

Hope it may help you

Hello, use meat peptone agar tat's it. A cheap solution is to boil some meat and then use bullion from it with some agar to make media.

Good luck!

viable count

Thank you soo much Sir@Prem Baboo

An OD of 2.6 would be quite high for E. coli unless you are using special media and aeration techniques, or baffled flasks etc. In a normal flask or culture tube I would expect an OD around 1.5-1.8. Also note that you can't actually accurately measure above 1.0 (assuming normal 1cm cuvette) unless you dilute before you measure.

However if your OD is actually decreasing significantly, this might be a sign of some type of lysis, but it probably depends on whether you are seeing slight decline or a significant decline. I would not worry about a slight decline but if significant then I would worry.

Kailash CHANDRA Srivastava and Shuraik Kader Thank you so much.

Abdelahhad Barbour Thanks for the idea !

is there any alternative martial of Hemin ? since it is hard to be found Saajida Akhalwaya

Hi,

After you grow the bacteria in liquid media, centrifuge them and save the cell pellet and supernatant. Find a solvent in which your secondary metabolite is soluble, but is not miscible in water (Butanol, ethyl acetate, chloroform, or dichloromethane are good places to start). Then:

1. Add the solvent to the liquid culture at a 1:1 ratio and shake at room temperature for 1 hr.

2. Allow the mixture to separate into phases (you can centrifuge the sample to speed this up)

3. Collect the solvent and evaporate to a workable volume.

4. Analyse your sample by LC/MS or thin layer chromatography.

You will likely get a large range of metabolites in the crude extract, which you can separate with a column if necessary. You can also do an extraction on the cell pellet using the same method, though you can use methanol or another miscible solvent because you will not have a large amount of water in the cell pellet.

Hello Minal Bhalerao

In case you wish to raise a secondary culture (in a minimal medium) from a primary culture (grown in a nutrient-rich medium, eg: Nutrient broth, Luria broth, etc.), you should wash your cells before inoculating them. This is done to remove the spent (used up) media from the primary cells or any metabolites or nutrients that would contaminate your minimal media.

Whenever we shift down the culture (transferring cells from a nutrient-rich to a minimal or nutritionally-low media), it is recommended that we wash the cells before inoculating them to raise the secondary culture.

Washing will not have any impact on the growth pattern. But, instead change/shift in media will cause a definite and notable change in the growth pattern. Shifting down would certainly extend the lag phase of your culture. If you wish to perform any growth-related experiments, using a common media for raising primary and secondary cultures is advisable. This would not extend your lag phase and your cells will begin to grow quickly.

Good luck!

Deepak Chand Sharma Thank you so much for your answer!

12/23/21

Dear Sayak,

I agree w/ Phil Geis' answer. Filtration will sterilize the oil. However, most oils are thick and viscous, so running them through a sterile filter will be time consuming. Autoclaving is a better way to go.

I hope this information helps you.

Bill Colonna Ames, Iowa, USA williamjcolonna6@gmail.com

Haoyang Zhou I suspected oxygen, too. I tried double agar overlay and it seemed to have solved it partially. However, not good enough. I think there is more to it than just oxygen. My bacteria was bloodborne pathogen so I am also thinking the environmenta pH may also play a role. In TSA, the microenvironment of even several bacteria may be changing enough to inhibit growth. However, in TSB buffering capacity is larger thanks to diffusion. I even considered of using blood and the buffer that are used to cultivate Bordetella pertussis, but it is a bit expensive for me currently.

Read above mention two papers, hope it will be helpful to conclude the importance of bacterial growth factors.

Aerosols generated e.g. by pipetting and (shared) centrifuges might pose a problem. Use designated sets of labware and establish a protocol to minimize contamination risks, e.g. by doing routine tissue culture before bacterial work, cleaning surfaces of the bench and frequently touched objects, as e.g. incubator doors) changing gloves, etc. Make sure to educate all people properly, esp. those who are new to the matter.

There are many tests, have you tried a simple means comparison test?

Cleaning the cells means centrifuging the cells and washing them with a simple buffer system (e.g., phosphate-buffered saline). The less components in the wash buffer the better. The problem with this, however, is that the metabolic composition of the cell changes pretty quickly and you are effectively stressing the cells by doing this. You also want to avoid heat or cold shocks so the buffer should be at the same temp as the growth media. Snap freezing the pellet in liquid N2 or dry ice after the wash helps preserve the metabolites.

The issue with the derivatization will be what in the buffer system also gets derivatized. Simplifying the wash buffer helps with this, but you should be aware if there is any carryover derivatives in the wash buffer. The concept of a control is more related to comparing the metabolome between growth conditions. LC/MS is not quantitative because of ionization differences between individual molecules. Therefore, when you are comparing LC/MS results between two culture conditions, all you get is a ratiometric measurement of what went up and what went down. You don't get an absolute concentration. You can only get to an absolute concentration by spiking samples, preferably with stable isotope versions of the metabolites that you want to track. see:

Hi Christian,

I suggest you to take just half of the volume per well of gel. In addition always run your gel between 70-80V range. One more thing i wish to add is monitor the buffer temperature, above 40 degree C may hamper the integration of gel subsequently resolution may affect.