Membranes - Science topic

I suggest reviewing the following paper:

Maybe check the blocking buffer? always use fresh made one or frozen stocks. Or does the Super-ECL reagent is fresh made? Or check the expose machine, do you use the correct filter?

you could also perform thermal analysis, like DSC and TGA to distinguish the grade and/or purity.

but if water can pass through it then it is obvious that product formed can also pass through it if we are doing any redox reaction.

Anyone who is expert in Reverse Osmosis? I'm still looking for an answer.

Since regenerated cellulose (RC) dialysis membranes allow water molecules to travel through them, it seems possible to use them to transport water for humidification without having water come into direct contact with the air. Let's talk about one way to make a dialysis bag or cartridge airflow humidifier for cheap:

Air-Misting System Modeled on Dialysis Technology:

Use a dialysis bag or cartridge with a large surface area to enhance water transport. Always keep in mind that a more significant surface area means greater efficiency.

The dialysis bag or cartridge must be submerged in a water reservoir. Maintain a steady level of water in the pool. The water level should constantly be above the membrane's surface for optimal humidification.

To avoid condensation, humidified air should be circulated in the dialysis bag or cartridge. A simple fan or blower will do the trick here. For optimal exposure and humidification, the flow should cover as much of the dialysis membrane's surface area as feasible.

Keeping a gradient in the water pressure across the membrane is essential for efficient water transfer. Creating a driving force for water molecules to migrate from the reservoir, across the membrane, and into the dry air can be accomplished by circulating dry air (low humidity) around the dialysis bag.

The rate at which water moves can be significantly impacted by ambient temperature. If possible, humidity rates can be increased by gradually warming the water reservoir.

Make sure you clean and maintain it regularly. The membrane could become clogged, or the water quality could decline over time. Consistent operation can be done by changing the water and periodically cleaning the system.

A cheap humidity sensor can be integrated into the system for advanced monitoring. This allows the output air's humidity to be tracked so the system can be fine-tuned.

Difficulties and Factors:

Water transport efficiency using RC membranes may be lower than that of more advanced Nafion-based devices. If we want to know whether or not this system can handle our unique humidification demands, we'll need to run some pilot tests.

Longevity of the membrane: It is unknown how long RC membranes will last when used for constant humidification. The membrane's effectiveness or integrity could decline with time.

As water vapour flows across the membrane, contaminants in the reservoir may become concentrated. Because of this, the performance of the membrane may degrade or become fouled.

RC dialysis membranes can achieve Humidification without air-water contact, making them a promising low-cost alternative. However, it is essential to be aware of the membranes' limitations and to optimize the system accordingly. Its performance may fall short of more expensive systems, but if well-designed and maintained, it can still perform adequately for some relatively inexpensive uses.

There is a recent study that considered CO₂ absorption into hybrid graphene oxide/MOFs, which might be useful for your perusal:

Hi Rizki!

I think your blot has improved significantly! I think I accidentally advised you to use PBS for this... please ignore that. Unfortunately, the problem with anti-phospho antibodies is that they are highly selective, and its a general trend so probing with it seems like a difficult task anyway. W.R.T. the difference in band intensities could be because of the way you treated your samples. While serine phosphorylation remains relatively stable post-heating, histidine phosphorylation if you're probing that, is really labile, and it is possible that you have this background solely because of low specific binding. I think going by the trend of your blots, reducing the concentration of your primary antibody further and increasing the incubation time might help you. as Esra Buber suggested, you may increase the concentration of BSA too for blocking.

One more weird thing that has worked for me while attempting to remove non-specific signals, is to wash the blot a couple more times after you're done with original washes, with TBS without tween. See if it works...

Thank you Paola Bernardo

Graphene oxide is an excellent building block for many membrane applications, including air dehumidification, water desalination, organic solvent mixtures pervaporation, electrocatalytic membranes.

But, it should be kept in mind, that:

1. Graphene oxide produced by different research groups could vary strongly in it's properties because of various flake size distribution, C/O ratio, synthetic and purification methods. So, when compare the data published by different groups, be careful.

2. Graphene oxide could be prone to ageing. It has dynamic structure, and GO-based thin films change their membrane properties with time. It's important to work with fresh GO suspensions and as-prepared membranes. The fresh GO suspensions should be stored in refrigerator, and avoid light exposure.

3. Because of ageing, it's of huge importance to carry out long-term monitoring of GO membrane characteristics (composition, flux, permeability, selectivity) with time (at least, for several months). To prove the stability of the membrane for practical applications.

What type of membrane distillation do you want to do? I have been working on direct contact MD and I do not use a spacer/mesh. The DCMD module has a rubber gasket on each side and the membrane is set in place by that and never sticks to the surface of the module. If you want/need to use the spacer/mesh, I suggest doing short experiments without it, with it on the permeate side and finally with it on the retentate side. After, you can analyze the system performance and characterize the membrane, and with that reach a better conclusion on the best approach. If your membrane has some type of support (I use a PTFE membrane with a PE support), the support faces the permeate side of the module.

Dear Reihaneh Abouei ,

Welcome dear Reihaneh.

Why not!

You can send me the SEM image and I will tell you the thickness. Be careful that the SEM image has to have a scale bar.

Best wishes

Dear Md. Mahmudur Rahman ,

Thank you so much for your response. I edited my question. My flat sheet membranes are fabricated from PVDF and are porous and hydrophobic.

These holes are only in some membranes even when no additives are used. However, I wanna use these membranes in the module and check their performance because a lot of materials are used to fabricate these long membrane sheets.

Probably I can use a special type of glue in a very small amount to cover these tiny holes. But what is that glue?

Or, probably, there are some other solutions.

What type of plasticizers I can use in hydrophobic PVDF membranes?

Best regards

skim milkMuhammad Mansoor Shaikh Muhammad Ishaque 、您可以在普通实验室中使用哪些方法，如 BSA 或脱脂牛奶

increase the concentration up to 15%, thickness is also low(increase it) and after collecting dry in a vacuum oven

Jiawei Lu

It all depends on the technology that you have chosen with or without a support layer. With a support layer, the membrane will be stronger, but this layer can interfere with filtration and contaminate the resulting product, the filtrate. Membrane characterization is secondary.

Good afternoon Max,

According to my AI desktop, which isn't always as reliable as I'd like:

"The main difference between AEM (Anion-Exchange Membrane) and PEM (Proton Exchange Membrane) is the ion that they transport. PEMs transport protons, while AEMs transport anions.

The main technical difference between AEM and PEM electrolyzers lies in the type of membrane used and the resulting electrochemical reactions that occur."

All I can think of is that your primary antibody against SMAD2 is not specific enough? (i.e. it is binding non-specifically to other proteins, given than you see signal all over your lanes). Are you able to try a different primary antibody against SMAD2? The best thing to do is to check the literature associated with the antibody you are buying; most of the time, companies will link you to different papers which have published data using that specific antibody.

Maybe another thing you could try before buying a different antibody is to dilute the SMAD2 antibody further? Sometimes if your antibody is too concentrated it can bind more non-specifically and lead to unexpected bands.

A dialysis membrane of 1 Kilodalton is not reusable for the same sample. Dialysis is typically a one-time use procedure to avoid contamination. The size of the carbon dots and the membrane's molecular weight cutoff determine whether a dialysis membrane is suitable for large quantity carbon dots filtration. Other filtration methods, such as ultrafiltration or tangential flow filtration, may be more efficient for large-scale purification.

Avoid to heat that much, it may lead to aggregate formation. In general, incubation in Laemmli buffer (containg SDS of course) at 37°C for one hour or so must be enough to give correct results on migration and Western Blot.

In Microbial Fuel Cells (MFCs), both Proton Exchange Membranes (PEMs) and Cation Exchange Membranes (CEMs) can be used as ion-selective barriers to separate the anode and cathode compartments. The choice between PEM and CEM depends on the specific MFC design, operating conditions, and the desired performance.

Performance Comparison: The choice between PEM and CEM depends on the specific application and design considerations of the MFC. In general, PEMs tend to offer better performance in terms of proton transport and electron selectivity, which can lead to higher power output in air-cathode MFCs. However, in certain configurations and operating conditions, CEMs may provide suitable performance while offering advantages such as improved durability and resistance to biofouling.

It's important to note that ongoing research and development in the field of MFCs may lead to advancements in membrane materials and design, providing even better performance and efficiency in the future. When selecting a membrane for an MFC, it's crucial to consider the specific requirements and goals of the MFC application and conduct appropriate testing and evaluation to determine the most suitable membrane type for optimal performance.

GFP is a naturally occurring fluorescent protein that can be genetically engineered into bacteria and other organisms to track and visualize protein localization or expression. In bacteria, GFP can be expressed as a fusion with other proteins of interest to study their subcellular localization.

Dear Dr. Maral Tabrizi

RIPA lysis buffer which contains a combination of both ionic and non-ionic detergents results in the highest number of identified peptides and proteins.

Yes, you may lyse EVs in an appropriate volume of RIPA lysis buffer. You may add protease and phosphatase inhibitors at the time of lysis.

You may use the following protocol.

You may use the following inhibitors in the RIPA lysis buffer at the time of lysis.

1 mM Sodium orthovanadate,

50mM NaF,

5 µg/ml Pepstatin,

5 µg/ml Aprotinin,

5 µg/ml Leupeptin.

Lysis may be performed on ice for 30min and placed in an ice-cold sonication bath for 30 s. This step may be followed by a gentle mix on ice for 15 min followed by centrifugation at 15000 g for 10min. Discard the pellet.

You may add 1 volume of 100% (w/v) TCA to 4 volumes of the sample, vortex and incubate for 5 min at 4°C. After centrifugation at 15000 g for 5min at 4 °C, the supernatant may be discarded, and the pellet may be washed with cold acetone. Centrifuge at 15000g for 2 mins at 4°C. Discard the supernatant. Repeat the washing step but this time using 80% cold acetone. Discard the supernatant. Air-dry the pellet (proteins) for 3 mins. You may dissolve the protein in the buffer of choice.

If you wish to use the micro-BCA assay, you may add 5μL of sample to a 96-well microplate followed by 100μL of BCA reagent. Mix plate thoroughly on a plate shaker for 30 seconds. The plate may be incubated in the dark for 30min at 37 °C. The absorbance may be measured at 560nm, and protein concentration may be determined from a BSA standard curve.

Regards,

Malcolm Nobre

Transwell inserts are permeable support device meant for the study of cell-lines to study angiogenic inducing or inhibiting effect on the migratory response of epithelial cells. I would request you to go through the publication of Sip CG, et al. Lap Chip 2014; 14:302-314; where it had been described with method compatible with conventional cell culture.

Dear Makayla,

I am not sure if you can quantify the amount of fouling based on FTIR alone. But let's discuss your issue a bit and hope I get your point. for any analytical analysis, we need a calibration standard. Let's assume you have a known feed solution concentration and you are using cross-flow flow filtration, after you stop the filtration, the concentration of your concentrate and permeate can be determined, so assuming the rest caused fouling. Remember that the volume of your initial feed, and permeate and concentrate are important to verify the mass balance. At this point, you can assume that the fouling occuoccurredcross-flowred evenly and calculate the amount of foulant per area of the membrane.

Another method is also, to cut 3 pieces (known area) of the fouled membrane, and then put that in 3 separate glass tubes (triplicate) and add a known amount of solvent that can dissolve your foulant. Afterwards, use an analytical instrument that can determine the concentration of your foulant which you can easily convert to gr based on the known added volume to the tube. Calculate the average and divide per area.

Last but not least, you can start with different known concentrations of sodium dodecyl sulfate in buffer solutions and do the same procedure that I explained and further also use FTIR and connect the concentration to the intensity or area under specific peaks if the peak is specific to your foulant and not the membrane. Now you can have a calibration curve that connects the ftir and concentration of foulant per area of the membrane. Just remember the time of filtration and pressure, temperature, flow velocity, (turbulent flow), pH and other important factors should be constant.

In the case of real-time monitoring of fouling using Raman spectroscopy, you can read the article of my colleague and maybe get some hints.

In one of my papers, I was also trying to identify the amount of vanillin adsorbed on the membrane surface after my experiment and I explained the methods there, which is similar to what I have learned from my colleague in the abovementioned article.

Hope my explanation is clear and I got your point correctly.

Yes

Evan Kerek and Saddah Ibrahim thank you for your answers

This paper comes to mind (not electrodialysis, but electrolysis): https://pubs.rsc.org/en/content/articlehtml/2020/ee/d0ee02173c

To simulate evaporation through porous media with the evaporation-condensation method in Ansys Fluent, follow these steps:

Set up your porous media: Define the porous zone in your domain where you want to simulate evaporation. Assign the appropriate porous media properties, such as permeability and porosity.

Enable the evaporation-condensation model: In Fluent, go to the "Models" panel and enable the "evaporation-condensation" model.

Define phase change settings: Set the appropriate parameters for the evaporation-condensation model, such as the evaporation rate, condensation rate, and heat transfer coefficients.

Specify the initial conditions: Define the initial temperature, pressure, and other relevant properties for the domain and porous media.

Set boundary conditions: Apply appropriate boundary conditions, such as inlet and outlet conditions for the fluid flow and evaporation.

Initialize and solve: Initialize the simulation and run it to solve the fluid flow, heat transfer, and evaporation-condensation phenomena.

Post-process results: Analyze and interpret the simulation results to understand the evaporation behavior within the porous media.

Remember to consult Ansys Fluent documentation or tutorials for more detailed information on specific settings and inputs relevant to your simulation

Yes, of cause you may try to run UV-vis and try to detect the SPR peak of AgNPs in the range of 400-450 nm

Hello Smriti Chand

1. Yes, you are right. B-actin is also present in the nucleus, as a component of chromatin remodeling complexes. You may use α-tubulin as a marker for the cytoplasmic fraction.

The soluble α/β-tubulin is considered to be a reservoir of subunits that are available for microtubule polymerization in the cytoplasm, but there are reports supporting the presence of tubulin in the nucleus of cultured animal cells. The reason for nuclear accumulation of tubulin, which does not occur under normal circumstances, is a process of pathophysiological significance and may represent a defense mechanism against stress or malignant transformation. This is the reason why nuclear tubulin may be detected only in cancer or in transformed cells.

2. Calnexin is highly abundant in ER as well as the outer nuclear membrane. It has also been found at ER-mitochondria contact sites. But the possibility of contamination of nuclear fraction with membrane fraction cannot be ruled out. Nevertheless, you may try caveolin-1 for the membrane fraction. Caveolin-1 is a plasma membrane protein that has a fundamental role in the formation of caveolae and acts as a connective protein that organizes the macromolecular complexes on the cell surface which participates in intracellular signaling.

3. In addition to fibrillarin, you could use histone (H3) for the nuclear fraction. H3, one of the core histones along with H2A, H2B and H4 is known to form nucleosomes with nuclear DNA. It plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis.

You may try these markers.

Hope it helps!

Best.

Applying a uniform film manually using a wet film applicator can be challenging, especially with high-fluidity materials. Here are a few tips that could potentially help you improve your results:

Remember that practice is essential to develop a consistent technique, and even small adjustments can sometimes significantly impact the results. Don't be discouraged if you don't achieve perfect results right away; membrane preparation is an art as much as it is a science.

Dear Dr. Denny Kollareth

The protocol that you have shared could have potential negative effects on data quality. The process of stripping and re-probing can take a considerable amount of time and reagents. Moreover, the membrane stripping procedure may remove sample proteins from the membrane. Therefore, quantification of phosphoproteins following membrane stripping would not be ideal.

I would suggest that the above drawbacks from the traditional chemiluminescent method of detection could be overcome by using fluorescent western blotting which will allow you to detect both versions of the protein (multiplexing) on the same membrane.

Multiplexing your western blot typically require antibodies against the total and phosphorylated protein raised in two different species. Fluorescent secondary antibodies bearing two different fluorophores (with non-overlapping spectra) can be used allowing for multiplexing which means both phosphorylated and non-phosphorylated species can be detected simultaneously.

Another important aspect is to select antibodies which are highly specific for the phosphorylated protein state only. If the antibody is not specific, this can result in simultaneous detection of total protein and confound the interpretation of your results.

Other factors that you should consider while detecting phosphoproteins are:

1. The phosphoprotein must be kept intact. For this reason, protease and phosphatase inhibitor cocktails must be added to cell lysis buffer prior to use. These will help prevent proteins from being degraded and dephosphorylated. Keep the lysate always on ice.

2. You should avoid using milk for blocking the membrane because milk contains casein, an abundant phosphoprotein that can cause high background. Use BSA instead.

3. You should avoid using phosphate-based buffers like PBS as it can interact with anti-phospho antibodies, affecting the binding of the antibody to the target protein. Use Tris-based buffers, for instance, Tris-buffered saline with Tween-20 (TBST) instead.

4. Use proper controls.

Regards,

Malcolm Nobre

In relation to this topic, this paper nice paper really clarifies the situation:

Bayliss WM, The properties of colloidal systems. I. The osmotic pressure of congo-red and of some other dies. Proceedings of the Royal Society B 81, 269-286 (1909)

Hi Mai,

If the cells are small, firstly I recommend that you use smaller tip electrodes ~10 megaOhms. Secondly, once you form giga-seal, instead of suction, apply incrementing voltage or current steps (0.5 ms long at 1Hz) till you break the membrane, as all membranes will eventually break under such electrical challange (ie., electroporation). For details of the method, please see my previous publication for details about membrane electroporation, without using a dye. Once you determined the membrane breakdown potential threshold then you can use the same amount of voltage or current to succesfully break down membrane in a more controlled manner. I have developed this method in retinal whole-mounts a while ago, but I have been regularly using it in brain slices, cultured cells and human IPSC derived or organoid cells. Once you get used to it success rate is over 90%, and it is relatively simple.

best wishes,

Refik

Hello Mr. Alex. The most direct way to measure a skin layers (dense, non-porous portion) of a membrane is by direct observation with FESEM at high resolutions.

Alternatively, particularly for gas separation application, one can estimate the skin layer by backwards calculation from the equation to calculate gas permeance once the intrinsic permeability of the gas is determined from fully dense membrane. Example by Pesek and Koros (1994) [ ]

Dear Manisha,

In my opinion it is not a good idea to check xo-localization with a regular fluorescence microscope.

The staining of your proteins can come from two different focal planes, which means, proteins are not co-localized but look as if they were.

For membrane proteins it should be even more complicated , since you will have fluorescence from the membrane marker all over the cell.

Therefore, use a confocal microscope.

Good luck,

Sebastian

Jamoliddin Razzokov Ma'Mon Abu Hammad thank you for your responses, sir. I have taken note of the provided answer and will ensure its careful consideration. Thank you for your time and assistance.

I would put the dialysis membrane in a beaker filled most of the way to the top with the buffer, add a magnetic stir bar, place the beaker on a magnetic stirrer, and set the stirring speed fairly high to make sure the contents of the bag are thoroughly mixed, but not so fast as to endanger a collision between the membrane and the stir bar. The volume of the beaker should be as small as feasible to contain the dialysis membrane while still allowing it to stir, thereby maximizing the concentration of citral in the buffer.

If this would not provide a sufficiently high concentration of citral because of the large volume of buffer, another idea would be to put the dialysis membrane in a sealed tube, such as a 50-ml conical tube with screw cap, filled with buffer and put it on a shaker platform, shaking at the fastest rate you consider to be safe for the dialysis membrane.

A possible problem with this experiment would be if the citral is not soluble in the dialysis buffer.

Daniela Liebsch Thank you for your answer. Yes, the gel is totally fine. I always activate the gel for total protein and everything seems normal. I will try to troubleshoot by the possibilities you mentioned

You might also try running separate, replicate gels, one for phospho- and one for total protein. Instead of stripping and reprobing.

Also, are you 100% confident in the specificity of the phospho antibodies? Is it possible the bands for pSTAT1 and/or pSTAT3 are really aomething else?

Armando Eduardo Fernandez

Yes, it does. There are several tips you can do:

1. As Mr. Manuele mentioned, check the pore size.

2. Air-dry the membrane after blotting.

3. Fix your membrane after blotting and before blocking (gonna add a reference).

4. Some labs use two membranes instead of one.

5. Adjust the time, current and voltage of blotting.

6. Make sure that you use 20% methanol in the blotting buffer (if you do not want to blot HMW proteins at the same time).

Pls refer to the following article for more information about fixation:

10.1016/0003-2697(89)90634-9

@Foroogh Khodadadi

Thank you for your explanation

We know that PEG and PVP have different molecular masses. What molecule do you recommend to use for these substances?

Your idea presents an interesting perspective on explaining consciousness and its relationship to neural activity. It incorporates the concept of frequencies associated with sensory stimuli and suggests that the brain acts as a "compiler" to translate these frequencies into conscious experiences.

While your analogy to programming and the idea of the brain decoding specific codes is intriguing, it's important to note that the nature of consciousness is still a topic of active research and debate in the field of cognitive science. There is currently no widely accepted theory that fully explains how neural activity gives rise to subjective experiences.

The relationship between neural processes and conscious experiences is a complex and multifaceted phenomenon. It involves not only the transmission of signals across neuronal membranes but also the integration of information from various brain regions, the role of attention, memory, and the influence of cognitive and emotional factors.

While frequency processing and neural coding are areas of research interest, it is important to consider that consciousness likely involves more intricate mechanisms than a simple decoding of frequencies. It is likely to involve the dynamic interactions between neurons, the emergence of complex networks, and the integration of information at various levels of processing.

Overall, your idea provides a thought-provoking perspective, but it is essential to approach the study of consciousness with an open mind and consider multiple perspectives and theories. Ongoing scientific research continues to explore the nature of consciousness, and future discoveries may shed more light on this fascinating topic.

Dear Zahra Khezri ,

Could you please add a photo of your synthesized membranes? Then, it would be easier to understand what is happening there.

Thank you so much

Best wishes

You can try with 40 - 60 minutes transfer adn blocking 2-3 hour

In a membrane-packed bed reactor, the presence of a membrane introduces permeation, which affects the fluid velocity distribution within the reactor. To model the effect of permeation on the velocity, you can consider the concept of mass conservation and use appropriate equations that account for both convective flow and permeation. One commonly used equation is the Continuity Equation. Here's how you can incorporate permeation effects into the velocity modeling:

∇ · (ρu) = 0

where:

∇ · (ρu) + ∇ · (ρ_pu_p) = 0

where:

This equation combines the convective flow (first term) and the permeation flow (second term).

It's important to note that the modeling of membrane-packed bed reactors involves additional considerations beyond just velocity, such as concentration profiles, reaction kinetics, and mass transfer limitations. Depending on the complexity of your system and the specific phenomena you want to capture, you may need to incorporate additional equations or models to accurately represent the reactor behavior.

Consider consulting literature related to membrane-packed bed reactors or reaching out to experts in the field for guidance on specific equations, correlations, or modeling approaches that would be most appropriate for your system.

Asif Khan Thank you very much for the detailed explanation. Definitely helped me.

Was it a strong signal? Remember, it's a reaction catalysed by the HRP you've stuck to your membrane, and if you've stuck a LOT of enzyme activity there, the reaction will proceed rapidly in that specific location.

For really intense signals its not uncommon to see "negative" blots, where the background levels of luminescence are visible on the film, while the actual signal is a blank void (having burned out all the ECL reagents before the film can even be applied).

You can fix this by...well, loading less on your gel.

My apologies, Burcu Yener Ilce . I focused on the problem and potential solution and didn't pay total attention to your explanation. To be clear, I the person I chatted with mentioned he air-dries the slides in the hood. I hope you find a solution soon. I should try it on Monday, but I am doing cryosectioning.

Chitosan is mainly hydrophilic. If it shrinks, maybe your environment's relative humidity RH % is not too high, or the temperature is too high. I recommend looking for sorption isothermal graphs. Good luck.

I agree with what the previous colleagues said. However, I think the crux of the matter is that your selection layer has some defects. Just like when the bathtub is filled with water, the water will all slip away from the downpipe. This question has run through the pre-experimental phase of every work I've done since I started doing membranes. Until one day, I suddenly wanted to understand this matter in a dream. Actually every material, every polyamide layer, is different, just like there are a thousand Shakespeares for a thousand people who read Hamlet.So, when you make each membrane, if it is what you said, I think you will get all kinds of strange and irregular results. In fact, for the synthesis process of each polyamide layer, I think that not only following the literature, but finding a method similar to interfacial polymerization, I think that the polyamide layer can be synthesized with peace of mind. As several colleagues said before, if it were me, I would have a high probability of not succeeding if I followed them strictly. Therefore, I think the most important thing for synthesizing a defect-free selection layer is to understand the properties of each component, pay attention to the details that are not mentioned in the literature, and boldly repeat the experiment. Just like some selective layers are not highly cross-linked, if you blow it with nitrogen with too much pressure during the drying process, it is bound to have defects. Therefore, dear colleague, I suggest that you use the slowest speed in history when you make a membrane next time. After each step is completed, think carefully about what contribution this step has made to the synthesis of the selective layer. What are the results. In this way, step by step, even if you are not successful, you will make new discoveries, and then you will be closer and closer to success. Some people attribute this process to "manipulation", but I am more inclined to sum up the experience in a large number of experiments, just like the old saying "practice makes perfect（熟能生巧）".

Hope my seemingly lengthy conversation can solve your problem. In the meantime, any comments from colleagues are welcome. good luck!

Difficult to guess what's going wrong. Have you tried other stripping methods, like 10mM or 100mM NaOH for a few minutes? Beware, too concentrated NaOH is dissolving NZ membranes quickly.

Thank you Dr. Liger and Dr. Stolz.

For anyone interested, I've found the site below to be helpful in visuallizing transmembrane domains.

I'm still in the troubleshooting phase of this issue. I can see there are multiple transmembrane domains in my protein, although I don't know if any of these can be considered a signal sequence or not. Never mind whether or not it is cleavable...

I figured expression in yeast would be appropriate as a previous study injected capped-RNA of the coding sequence into frog eggs and produced functional transporters. I just need to re-sequence my expression vector maybe..

This depends on the type of cancer you are investigating and the tissue from which the FFPE sections are made. As mentioned, e.g. T-cell lymphomas show cytoplasmic CD3 staining.

Try to figure out in which tissue/cell type you see the cytoplasmic CD3 staining.

By following these steps, you will have diluted your crude membrane samples to a concentration of 1 mg/ml and stored them as 100 μl aliquots for future use in the 35sGTP gamma assay.

Found it. Here are the lines that can display the cholesterol on VMD. In the display box just put: "resname CHL1" then create a rep and type "lipid and not water" and change the color IDs to be able to distinguish them. To see the resname for Cholesterol, open your PDB file textfile and search for CHL in the residues (usually at the bottom). I have attached pictures:

Hi Rebecca,

It is depend on how you cut the membrane. General rule for the WB full blot is that it contain ladder on the side (so that the protein size can be verified), have the internal control (so that the protein expression can be relatively quantified), and the blot exposure is optimised (so that there is not over- or under exposure that hinder the unspecific bands). While you can have different crop in the manuscript, but often reviewer will need to to have the full blot in the supplementary section so that they can verify the protein data. In other word, you can have the cut as it feed your work, but you will need to assembly them into a full blot so that the reviewer can assess it.

Probe the whole membrane and use a "rainbow" western for several targets. Cutting the membrane does not save sample, as you need a certain amount in a band for detection. It also does not really save primary antibody, as that solution can be used many times (use 0.1% BSA in PBS as solvent with timerosal as preservative, develop blots at 4 degrees o/n). But you preserve the information about antibody specificity (that you get a single band).

what if I dont want to apply tmp on the FO?

Arunima Verma I have the same problem. Did you find how to solve it?

Yes!! Very nice tube.

I can't answer your question because I study only advanced compound semiconductors with SEM.

Just because the total protein expression was similar between samples and heart vs kidney doesn't mean the expression of a particular protein will be stable. You always should validate whether your loading control / reference protein expression is stable across your experimental groups.

Hi,

I am getting the same error "FATAL ERROR: DIDN'T FIND vdW PARAMETER FOR ATOM TYPE ON3." There is no ON3 atom in the pdb and psf files.

Any solution how to troubleshoot this?

In reply to your question:

"How to choose suitable buffer for liposome preperation?"

Gokulakrishnan Manimaran

We can chose from a huge list of buffers of alternative hydration media based on the application we aim for.

From the Reference (listed below and attached):

" ....

Liposome hydration or suspension media can comprise any of the above-mentioned buffers or saline solution (0.9% w/v of NaCl in distilled water) or isotonic sucrose solution (9.25% w/v sucrose in distilled water).

...."

"... Alternative hydration mediums are saline or nonelectrolytes such as a sugar solution. For an in vivo preparation, physiological osmolality (290 mOsmol/kg) is recommended and can be achieved using 0.6% saline, 5% dextrose, or 10% sucrose solu- tion (24). The aqueous medium can contain salts, chelating agents, stabilizers, cryo-protectants (e.g. glycerol) and the drug to be entrapped. ...."

Mozafari, M. R. (2010). Nanoliposomes: preparation and analysis. Liposomes: Methods and Protocols, Volume 1: Pharmaceutical Nanocarriers, 29-50.

Nourhan S. Elkholy

A lot of different suggestions have been made. Perhaps the key message is to only ever change exactly ONE parameter when comparing images of different structures, as there are many (acc. voltage, current, dose, vacuum level, detector type, coating thickness, coating type...) that can influence what you see. Certainly 20nm of C coating can already start to cover many small details incl. holes that tend to be overgrown (you can buy holey C films of about 10nm thickness as TEM supports, which look a bit like the images here). Whether you use Au or C coating, my suggestion would be to coat with as low amounts as you can get away with to just avoid charging - I also assume the polymer itself will be beam sensitive so try low kV and smallest possible beam current, and then watch your specimen while scanning the beam - it may change while you watch!