Membranes - Science topic

Hello Thamizhiniyan, I used more blotting paper. I used 3 paper up and 3 down to make a western blot sandwich and also I kept cold ice packs in the transfer tank during the transfer process. I hope it helps.

problem in northern blotting is often sample degradation by RNases (both endogenous to the sample and through environmental contamination), which can be avoided by proper sterilization of glassware and the use of RNase inhibitors such as DEPC (diethylpyrocarbonate)

Northern blots are used to detect the presence of specific mRNA molecules. To do a northern blot, RNA is loaded into the wells of a gel, and separated according to size by electrophoresis. The RNA is then transferred to a membrane filter in a process called blotting.

Northern Blots: 0.05 fmol detection limit with near-IR RNA Probes.

To disrupt the secondary structure of RNA, either formaldehyde or glyoxal/DMSO (dimethyl sulfoxide) is commonly used as a denaturing reagent. Formaldehyde is simply added to the samples and gels, so that the method using formaldehyde is easier to run the gels than that using glyoxal/DMSO.

oh, thanks so much. I have used 14kda let me see how the results come out.

@ Timothy, I think the probability density function (PDF) is the most suitable method which determines the mean pore size along with the pore size distribution (PSD) of nano-filter membrane.

Dear Mariam,

It is advisable not to mix two primary antibodies even if they have requirement for same secondary antibody. Cutting the blot based on the molecular weight is one approach and the second one is to follow sequential incubation.

Good luck with your blots.

Best,

Sheethal Galande

PhD Fellow

AHF

AIG Hospitals

Exosomes are mostly negatively charged. Like all materials, exosomes spontaneously acquire surface electrical charge when brought into contact with a polar medium, such as a hydrophilic buffer. Therefore, the exosome surface will generally be negatively charged in such buffers.

Quantifying the quantity of CO2 adsorbed by the membrane requires multiple processes, which are important to examine CO2 capture simulation on activated carbon filters in a duct. First, employing activated carbon filters, an experimental setup or simulation model must be created to mimic the CO2 capture conditions inside the duct. To ascertain the activated carbon's relevant characteristics for CO2 adsorption, such as surface area, pore size distribution, and functional groups, characterization is essential. The relationship between the concentration of CO2 in the gas phase and the amount adsorbed by the filters is then determined via adsorption isotherm tests or simulations.

The amount of CO2 adsorbed is then determined by mass balance calculations that take into account the gas flow rate, adsorption capacity, and concentrations at the input and output. In addition, CO2 adsorption behavior under various operating conditions can be predicted using mathematical models or computational simulations; the quality of these predictions is ensured by confirmation against experimental data. This scientific methodology allows for the development of CO2 capture applications for increased efficiency by providing data on the CO2 capture performance of activated carbon filters in ducts.

Here are some concise tips to improve the quality of bands in your Percoll gradient:

1. Optimize Percoll Concentration: Experiment with different concentrations to find the optimal gradient for separating the basolateral membrane fraction.

2.Sample Preparation: Use effective techniques like homogenization or sonication to isolate the membranes efficiently.

3.Starting Material Quality: Ensure high-quality cells or tissue free from contamination.

4.Centrifugation Parameters: Adjust speed and duration to optimize separation.

5.Marker Proteins: Include specific markers to confirm the presence and purity of the fraction.

6.Gradient Fractionation: Collect and analyze fractions separately to locate the basolateral membrane fraction.

7.Quality Control: Use assays to confirm membrane protein presence and fraction purity.

8.Consult Literature: Look for optimized protocols and techniques in relevant research papers.

Obtaining an exhaustive list of all membrane receptors and passive ion channels involved in metabolite transport for a prokaryotic organism can be challenging due to the complexity and diversity of microbial transport systems. However, there are several approaches you can take to compile such a list:

Mr. Kaushik Shandilya, first of all, thank you very much for your advice and for taking the time to reply.

Thank you Sasikala!!

Hello Preeti Rathi

Transwell pore size should be chosen based on cell type and size. Generally, the selected membrane pore size should be smaller than the cell diameter when in suspension. You may perform a pilot experiment to determine the appropriate membrane pore size, which allows sufficient cell migration toward a chemoattractant while minimizing random cell migration without chemoattractant.

Generally, most leukocyte cell lines have a diameter of 10–15μm and will require a 5 or 8μm pore diameter. However, smaller cell types, such as primary lymphocytes, range between 7μm and 10μm in diameter and so may require a smaller pore size of 3μm to prevent high levels of cell migration in the absence of chemoattractant. Other cell types like fibroblast cells (for instance, NIH3T3) or cancer cells (for example, MDA-MAB 231) would require 5μm pore size.

So, I would suggest for PBMCs use a smaller pore size of 3μm since the average mean cell diameters range between 7μm - 10μm.

Best.

Do the NCM blocking before immobilizing the protein on it. Block the NCM with same buffer with optimization of BSA and Tween-20 (Increasing or reducing the percentage of concentration) and if you feel it decreased your sensitivity the instead of PBS, use 10mM Phosphate Puffer (KH2PO4). I hope it will resolve your problem.

The three pairs of dots in the corners are your reference controls. You should normalize the intensity of the other pairs to those.

In regards to ROI selection, every dot should have the same size in this blot. The control dots are overexposed and have bled into adjacent pixels, so I wouldn't just use the size of the largest dot for your measurements. The pair of dots near the middle of the top image looks to be the proper size, so I would recommend those as the "ideal" area. However, you can also use the integrated signal density (equivalent to the mean intensity * area) to deal with differently sized ROIs.

I recommend using the Licor Image Studio (https://www.licor.com/bio/image-studio/, the Lite version is free to use) for densitometric quantification. They make it simple to do local background subtraction and make it easier to assess the shoulders of your ROI.

ok! I will check thank you for your time.

Shell calcium was determined by the Ion Conductively Plasma (ICP) device.

ASTM. American Society for Testing and Materials. Water and environmental technology. Annual book of ASTM standards, USA Sect 11(11.01 and 11.02) West Conshohocken. (2002).

In terms of removing the shell membrane, it's simple to accomplish using a clip or by hand.

Dear Mohamed Selim ,

I think it might be a problem to get these membrane bound proteins into solution. I assume you are spinning down the cell debris after lysis? I do think that there is most on the CDH5 and Claudin going. Is there SDS within you lysis buffer? You might want to take a look in the literature searching for soluble (cytoplasmic/small vesicle) and insoluble connexin (membrane bound) lysis buffers.

Best wishes

Soenke

Dear Hazel Anne Cledera, you can use AFM to compare surface relief and groups. Surface tension or surface free energy experiment also witnesses on any possible changes on the surface. My Regards

Может быть поможет метод полива и сушки на стекле?

Dear Muhammad Younis ,

Thank you for your answer.

I got the result after hybridate with an antibody, i tried ponceau s again after that, the problem disappeared for some reason and the membrane looked normal. my supervisor asks me to do an overnight transfer, it seems 1h 100v does not work. Philippe Paget-Bailly

I usually air dry the membranes after dipping in methanol and seal it in a plastic bag at -20 degree celsius.

The Human Red Blood Cell (RBC) Membrane Stabilization Assay is a commonly used method for assessing the anti-inflammatory activity of substances. It involves measuring the ability of a substance to prevent hypotonicity-induced hemolysis or stabilize RBC membranes. While it is a widely used assay, its reliability depends on the specific context and the nature of the substance being tested.The assay provides an indication of the potential anti-inflammatory effect by evaluating the substance's ability to protect RBC membranes from disruption. However, it's essential to note that the correlation between this assay and in vivo anti-inflammatory effects may vary. The relevance and reliability of the assay depend on the specific mechanisms of inflammation involved and the intended application.

Just spot the RNA on a dry nitrocellulose membrane and let the RNA/spot dry. Then crosslink it.

Some possible reasons for seeing extra bands are (1) insufficient blocking, (2) an insufficiently specific primary antibody, (3) too high a concentration of the primary antibody, (4) insufficient quality of the antigen, containing multiple aggregates and proteolytic fragments.

Check by SDS-PAGE if the supposedly purified antigen runs as a single band using a heavy loading to see minor bands.

I want some suggestions for the covalent immobilization of aptamer on polymer membrane surfaces.

Alvena Shahid ohh. thank you so much!!!

Yes

Misbah Waheed Alternative methods include Surface Plasmon Resonance (SPR), Electrochemical Impedance Spectroscopy (EIS), Fluorescence-Based Techniques, Capillary Electrophoresis (CE), Microfluidic Devices, and Atomic Force Microscopy (AFM)

Dear Parinda Limprasert ,

Not sure what you are aiming for, but I guess a nice starting point is (when you basically only have the primary sequence) use: CCTOP (https://cctop.ttk.hu ).

CCTOP: a Consensus Constrained TOPology prediction web server

For way more tools, see https://bip.weizmann.ac.il/toolbox/structure/2d.htm and scroll down to “Membrane Protein and Signal Peptide Prediction”

Best regards.

Didier Poncet We did use NaoH and NaCl for denaturing the DNA fragments in the gel before transfer. We have taken enough care while setting up the transfer so that there is no direct contact between the paper towels and reservoir buffer. Thank you.

What do you mean with membrane grow? They should not expand much, but some may have a gel-like behavior.

But how does it measure the thickness if the membrane is not complete plain, thus a reference point will not help?

So far i used microscopy to measure the thickness. However, the reproducibility is not reliable. Using a micrometer might not have sufficient accuracy.

I had a similar problem, the second detection was very weak, so I tried to divided my samples by half (repeated loading into the gel in the same order), and then after the transfer I cut the membrane and each half was detected by different antibodies, it worked every time.

Thank you so much professor Emanuel cooper ,it really worked for me.@Emanuel

Hello,

Increasing flux in a Fertilizer Driven Forward Osmosis (FDFO) system involves optimizing various factors to enhance the rate of water transport across the membrane. Here are several strategies to consider:

By implementing these strategies and optimizing various aspects of the FDFO system, it's possible to increase water flux and improve the efficiency of water treatment or desalination processes.

Alvena Shahid... would u please give some other references regarding this matter. Thanks in advance.

Нельзя

Dear Shukai Zhu Is this what you are looking for?

Kunová N, Havalová H, Ondrovičová G, Stojkovičová B, Bauer JA, Bauerová-Hlinková V, Pevala V, Kutejová E. Mitochondrial Processing Peptidases-Structure, Function and the Role in Human Diseases. Int J Mol Sci. 2022 Jan 24;23(3):1297. doi: 10.3390/ijms23031297. PMID: 35163221; PMCID: PMC8835746.

Best regards.

strange... I would say that in your 4 first lanes you have a very strong signal that leaks on the gel ... when the signal is very stong it can makes white bands instead of black ones... did you make a spot with a pencil ? (inks are fluorescent )...

Dear Stephan Spangenberg,

Thank you for your reminder! We have confirmed that the band size was due to non-specific binding. However, upon reevaluation using a highly sensitive chemiluminescent substrate, we successfully identified protein bands at the correct size.

Additionally, in the case of the splenocyte sample, after stripping and reprobing, I observed phosphorylated STAT1 (pSTAT1) but not total STAT1 protein. Interestingly, B cell samples appeared normal, and it is challenging to explain this phenomenon. To address this issue, I am considering running separate, replicate gels instead of stripping.

Thank you once more for your valuable input!

Hi Debarchan,

You may make an inquiry at Alfa Chemistry, they offer you advices and kinds of good-quality chemicals.

Karanjit Kapila My young friend! You can crosslink polyvinyl alcohol macromolecules with boric acid, for example. How to do it? See the link.https://cyberleninka.ru/article/n/sinte3-granulirovannogo-gelya-polivinilovogo-spirta/viewer

I used to do dot blots with a vacuum. In thinking back, if I added too much sample (in my case purified cell wall extract) then the whole disk of material would sometimes float up during the wash step. More diluted samples didn't have this issue. Maybe that might help your samples too?

Ah, my dear friend Pablo Gonzalez, when delving into the intricacies of membrane technology and its impact on pollutant removal efficiency, one must navigate the labyrinth of zeta potential values with utmost precision. Zeta potential, the electrokinetic potential at the slipping plane of a particle or membrane, plays a pivotal role in this symphony of separation.

Allow me to shed light on this matter with the finesse of an artisan. The zeta potential, influenced by surface charge, is a maestro orchestrating the forces at play. Membranes exhibiting higher zeta potential values often boast enhanced electrostatic repulsion, discouraging the adherence of charged particles.

This ballet of forces directly affects the pollutant removal efficiency, as a higher zeta potential tends to fortify the membrane against fouling. The repulsion between the membrane surface and charged pollutants mitigates their deposition, preserving the membrane's integrity and sustaining its efficacy.

As for the removal mechanism, envision this as a courtly dance. The zeta potential, akin to a vigilant sentinel, guards the membrane's honor against the encroachment of impurities. The electrostatic forces at play create a barrier, allowing only the most worthy particles to traverse, while banishing the undesirable with a noble disdain.

In essence, my esteemed interlocutor Pablo Gonzalez, a membrane adorned with favorable zeta potential values stands as a bastion against the incursion of pollutants, demonstrating an elegant dance of selective separation.

You can try with biopolymeric active materials like CNC-GO based membrane.

For better understanding you can follow this article.

Also this particular article could be beneficial.

Fatma Mohamed Fouad Yes, when using primary antibodies with different IgG subclasses eg. IgG2a & IgG2b, you can choose donkey-anti-species IgG2a for the first target, and goat-anti-species IgG2b, as such.

If you are probeing with multicolor (like Li-COR) then you can apply the antibodies at the same time, which is highly recommended. If not, then you can probe the first target then shift to the next.

Hello Kaushik

Thank you for your contribution. One minor correction: actually ceramics tend to use more energy than polymerics. Ceramics can be run at higher linear velocity, with a corresponding improvement in flux and reduction in rate of fouling. However, the downside is higher pumping cost, hence higher energy.

Regards

Alan

A lipid extraction procedure quantitatively extracts cellular lipids in an undegraded state and uncontaminated with nonlipid components such as free sugars and amino acids. The two most conventional methods of lipid extraction are, namely, Folch method and Bligh and Dyer method.Enveloped viruses acquire lipid membranes as their outer coat through interactions with cellular membranes during morphogenesis within, and egress from, infected cells. In contrast, non-enveloped viruses typically exit cells by cell lysis, and lipid membranes are not part of the released virions. Viral envelopes consist of a lipid bilayer that closely surrounds a shell of virus-encoded membrane-associated proteins. The exterior of the bilayer is studded with virus-coded, glycosylated (trans-) membrane proteins. The main component of the viral envelope is the host-derived lipid bilayer. The precise composition of this lipid membrane varies, as different viruses acquire their envelopes from different cellular membranes. The virus envelope is known as a capsid. Capsid protects the genetic material of the virus during the entire life cycle of the virus.these issues, the multi-omics sample preparation technique MPLEx (metabolite, protein, and lipid extraction) is developed to partition a single sample into three distinct parts (metabolites, proteins, and lipids) for multi-omics analysis, while simultaneously inactivating MERS-CoV by solubilizing and disrupting the viral envelope and denaturing viral proteins.

put it professional equipment manufacture company with blueprint. Once you start trading, it will become easier afterward. in your country, are there any metal processing companies that only specialize in manufacturing?

Thank you so much Chian Yong Loh

Hello

I think the deep coating method under sonication can give a good answer. In this way, put a part of the support membrane in the GO-TIO2 solution and at the same time keep it in the ultrasonic bath for a certain time. This makes it possible to provide proper coating and particle dispersion at the same time.

Nice SEM photo's!

It looks in the SEM magnification that both SEM magnifications are not the same? The main reason for the different between the two SEM is the fact that the coating thickness is different and the type of coating is different.

I understand that you are coating the filter in order to be able to SEM them as they need to be conductive. Try to use a high power microscope ~ 1000x just to observe how the filter looks. You may learn something.

Hello Quynh Pham

The protocol seems to be okay except for some modifications.

1. Try optimizing the LPS concentration and the time point for induction.

2. Try to reduce the concentration of secondary antibody. Use of too high concentration of secondary antibody may result in non-specific binding to proteins other than the protein of interest. Non-specific binding can occur with any protein. Thus bands of higher and lower molecular weight may be observed. Secondary antibody in the range of 1:15000 -1:20000 could be used. It’s a good practice to titrate antibody concentrations, thereby optimizing the signal to noise ratio. You may run secondary antibody control without primary to confirm specific binding of primary antibody.

3. Has the sample been completely reduced? Incomplete reduction of sample can lead to bands containing high order species which appear as bands at unexpected sizes or as smears. Use fresh reducing agent such as BME or DTT in sample loading buffer and boil in SDS (5-10 minutes).

4. The possibility of sample contamination may be another factor which is less likely to happen. Bacterial growth in running buffers can lead to unexpected bands on the gel. Always handle gels wearing gloves and use fresh buffers each time.

Hope these may help!

Best.

Dear Jakub Hryc In the similar question asked here https://www.reddit.com/r/botany/comments/17p1ehs/change_lipid_component_from_galactolipids_to/ there is a good reference to https://academic.oup.com/pcp/article/59/6/1128/4990989?login=false where the message is not so much that phosphate is limited (which is sheer nonsense because all (cell)membranes of animals, included us humans, and most of the bacteria are constituted of phospholipids) but “these two lipids [the galactolipids MGDG and DGDG] are important for maintaining chloroplast morphology and for plant survival under abiotic stresses such as phosphate starvation and freezing”

Best regards.

Could your problem be with the color metric solution? Are you using HRP conjugated ABs? Is your colorization agent DAB? If so, check the age of it. They do go bad over time and it's best to store them in the freezer when not using them.

Thank you Tushar Patil

Your answer is more informative

Dear Lira,

Can you elaborate the transfer parameters you run? Although you ruled out the possibility of insufficient blotting, I still believe that the problem is there. This is because it is not uncommon to face such a problem in semi-dry blotting

Two things spring to mind. One is that the protein has a natural proteolysis site near the C-terminus which is resulting in most of the protein being processed to what you see. The second is that the actual clone you are using for expression has picked up a mutation so that protein translation is terminated or frame-shifted near the C-terminus. You can check the latter by either sequencing plasmid from the exact clone you are using for expression, or checking that an independent clone/transformant shows the same behavior.

Have you tried some negative controls?

1. Stain an unused insert to see if it's a bad batch of inserts.

2. Stain culture inserts after incubation with your culture medium components: something in DMEM or your FBS Is a adsorbing omto the insert.

if those are clean then then I would suspect deposition/secretion of material by your cell line.

I need free standing PTFE film for hydrogen permeability measurement.

I tried many times to get free standing PTFE film, but I am unable to getting.

I used PTFE dispersion solution and I used spin coater, I cured at 347 Oc temperature.

Block regent like skimmed milk, if it didn't dissolve well, black dots would appear. use a stir equipment, then filtration the block regent.

As far as I know, ZView only analyses data already collected from potentiostat/galvanostat equipment, so the measurement has already been finished before it is opened on ZView. Since a current overload condition is something that happens during a measurement, I cannot see how ZView would report a current overload. There must be another software application controlling the hardware that performs the measurement - could you provide a screenshot or photo of the overload report that you've received?

I have been working with Streptactin-HRP to recognize TwinStrep tagged proteins. I did trials using BSA or milk to block (although they do not recommend using milk to block membranes as skimmed milk contains biotinylated proteins ) and no difference was found comparing these two for my experiments.

What I do after transfer (semi-dry system):

1. Stain with Ponceau to check the transfer and then, wash with 1x TBS-T (0.02% Tween) until dye is removed.

2. Block with a solution of 5% skimmed milk (powder) in 1x TBS-T for 1hour on an orbital shaker.

3. Wash 3 times for 10min each wash with TBS-T

4. Incubate the Streptactin-HRP (I was using a dilution of 1:50.000 IBA lifesciences in TBS-T) overnight in the cold room on an orbital shaker.

5. Next morning, wash 3 times with TBS-T (10min each wash).

6. Develop by adding ECL solution, incubate for 1 min and acquire image with iBright Thermofisher or X-ray films.

Normally I get very clean membranes with sharp bands, when the amount of the protein of interest was low, I could see some background from the membrane but nothing crazy. Looking at your membrane, there are patches that are very dirty, make sure when washing the membranes to add enough TBS-T to cover the membranes and avoid folding or bending the membrane, treat the membrane carefully and always use tweezers to work with the membrane.

Hi Matías,

You may make an inquiry at Alfa Chemistry, they offer you advices and kinds of good-quality materials.

Yao Xinyi Le Chang Anupriya Bandyopadhyay Thank you all for the kind answers!

I did once again and it worked really good!! But I changed not only the TBS-T, but also the transference configuration and I used the antibodies already diluted. :)

Dear Ahmed,

perhaps the following study, their references and publications that cite this study are helpful for your question:

Good luck.

Martijn

To be more specific, glass frits with considerably smaller pore sizes (down to the nanometer range) are commonly used in commercial reference electrodes, while frits with larger pore sizes are used in apparatus designed for accurate emf measurements in a potentiometric cell. The former application of porous frit junctions is analyzed by M.P.S. Mousavi et al. Anal. Chem. 2016, 88, 17, 8706-8713 As examples of the latter application, the evaluation of the liquid junction potential in a cell with frits of G3 porosity (E.E. Johnsen et al. Z. Phys. Chem. 1990, 168, 101-114) and the standardization of pH reference materials in a differential cell with the frit of G4 porosity (F.G.K. Baucke. J. Electroanal. Chem. 1994, 368, 67-75) can be mentioned.

Best regards,

Rouvim Kadis

Thank you

Building a simple PEM (Proton Exchange Membrane) electrolyzer is a valuable project, and creating a suitable proton exchange membrane is a crucial part of the process. Here's a suggestion for a synthesis method to create a proton exchange membrane using readily available materials:

Materials You'll Need:

- Sulfonated Polysulfone (SPS) or Poly(ether sulfone) (PES) as the base polymer

- Concentrated Sulfuric Acid (H2SO4)

- Deionized Water (DI water)

- Solvent (e.g., Dimethylformamide or DMF)

- Glassware and lab equipment (e.g., round-bottom flask, magnetic stirrer, vacuum oven)

Procedure:

1. Prepare the Polymer Solution:

- Weigh the Sulfonated Polysulfone (SPS) or Poly(ether sulfone) (PES) polymer and add it to a round-bottom flask.

- Add an appropriate amount of solvent (e.g., DMF) to the flask to dissolve the polymer. Stir the mixture until the polymer is completely dissolved.

2. Sulfonation:

- Slowly and carefully add concentrated Sulfuric Acid (H2SO4) to the polymer solution under stirring. The amount of H2SO4 will depend on the degree of sulfonation you desire, but typically, a small amount is sufficient.

- Continue stirring the mixture for a set period (e.g., several hours) at an appropriate temperature to ensure proper sulfonation. This step introduces the sulfonic acid groups into the polymer structure, making it proton-conductive.

3. Quenching:

- After sulfonation, quench the reaction by adding a large volume of deionized water to the flask. This step helps neutralize the excess sulfuric acid and stops the sulfonation process.

4. Membrane Formation:

- Pour the resulting solution onto a clean glass surface to form a thin film. You can use a glass rod to spread the solution evenly.

- Allow the solvent to evaporate slowly at room temperature or, if available, use a vacuum oven to accelerate the drying process. This will result in the formation of the proton exchange membrane.

5. Membrane Characterization:

- Once the membrane is dry, you can characterize it using various techniques, such as Fourier-transform infrared spectroscopy (FTIR) and proton conductivity measurements, to ensure it meets your desired specifications.

Please note that this is a simplified procedure, and the specific conditions and materials may need to be adjusted based on your laboratory's capabilities and the desired properties of the membrane. Additionally, always exercise caution when working with concentrated sulfuric acid and follow appropriate safety protocols.

AI answer

There are a few reasons why you might need to run acid digestion after membrane adsorption of Li+.

Whether or not you need to run acid digestion after membrane adsorption of Li+ will depend on your specific needs and the goals of your experiment. If you are unsure, it is always best to consult with an expert.

Here are some references that you may find helpful:

All I done collect 3 answers in one passage.

Good luck

Hi Subodh Sharma ,

So, I was able to isolate your stained cells with Colour Threshold function in ImageJ, though it's not perfect. The red region you are seeing will be measured when we proceed further for calculations. I have attached the screenshot. I hope this helps.