Science topic
Membranes - Science topic
Thin layers of tissue which cover parts of the body, separate adjacent cavities, or connect adjacent structures.
Questions related to Membranes
Hello all, in DPPC/DOPC/cholesterol ternary system, we observe ld-lo phase separation. My question is why cholesterol prefers to stay with DPPC rather than DOPC. Thank you.
For lateral flow test development, which membrane do you recommend most for conjugated pad? I currently use Whatman STANDARD 17 from Cytiva
I have made TL reagent by mixing antibody in 1X PBS containing 0.25% sucrose for striping similarly the CL which is anti species of TL and prepared using the above composition. While running the LFA, I can see both TL and CL increases linearly with my analyte concentration which was strange.
I have found out with my investigation that my test line antibody move during the running of the lateral flow and the TL antibody gets captured at the CL and forms the sandwich with the analyte there and hence I see the linear correlation with the analyte.
I need to know how can I immobilize my antibody firmly to the nitrocellulose membrane, such that it does not move and interfere with CL antibody.
Thanks,
Yogita
I've been running a simulation of thermal expansion. Two parts are defined and conected via TIE. one of the parts is surrouneded by a fixed (ENCASTRE) membrane surface, and the two are defined in general contact as pairs.
During thermal expansion I found the following warning:
"Some nodes involved in general contact have penetrated their tracked faces by more than 50.000 percent of the typical element dimension in the general contact domain."
The contact seemed to be ignored, as there is a deep penetration of the internal part through the outer membrane surface, despite the high density of the outer surface mesh.
The penetration is not only in the TIE area, but scatered all across.
Defining a Thickness surface to the parts in contact was impractical.
More info:
Abaqus version: 2017 Explicit.
TIE:
Hard contact
constraint enforcement method -
(Normal) Default. Allow separation after contact,
(Tangential) Fricttionless. 0;
Material properties:
Inner part:
*Material, name=RUBBER
*Density
1e-09,
*Hyperelastic, neo hooke, moduli=INSTANTANEOUS
200.,0.
*Viscoelastic, time=RELAXATION TEST DATA, nmax=5
*Shear Test Data, shrinf=0.3
1., 0.01
0.9, 0.4
0.82, 1.
0.72, 2.
0.56, 5.
0.44, 10.
0.38, 20.
0.3, 50.
*Expansion
0.001
Outer part:
*Material, name="Outer Shell"
*Density
1e-09,
*Elastic
9000, 0.29
How can I fix that?
I need to show the co-localization of my protein of interest in the membrane in HL60 round and small cells. So I took z-stack images to show the surface expression of the membrane marker that I used. But I don't know how to present my data for the paper.
Regarding Egg membranes as model cell membranes for diffusion experiments.
which is better, the synthetic membranes or egg membrane
Hi all,
I'm looking to conduct screening on a cell line of interest, using the Boyden Chamber Assay method, to identify migratory vs non-migratory phenotypes and then elucidate their matching genotypes.
I was wondering if anyone had any ideas of how to reliably separate the migrating cells on the lower transwell membrane, from the non-migratory cells suspended in the upper chamber?
Any ideas are appreciated...thanks!
Is there any explanation for the strange curve feature at the beginning of the AFM tip contacting the sample membrane? Is it that there is some dust attaching the AFM tip or any other reason? How to avoid it?
Hello, I have started working with Western blot recently and have difficulties getting the beta-actin band. I recently tried the steps for getting a beta-actin band from the intestinal sample of mice. I mentioned it here.
1- 10μl or a maximum of 20μl of protein into each well. Start at 100 V for 1-1:30 h.
2- Transfer protein from gel to membrane (make sandwich) 100V, 1:30h
• PDVF membrane; 1 min in methanol 100% for activating. However, I only get bands on the membrane after staining but no bands after the antibody-blocking steps.
I would be so happy if you could suggest me t how I can solve this problem.
Hi there. Have anybody noticed that the membrane time constant values in membrane test are in rage of micro-seconds (hundreds of microseconds but rarely more than few milliseconds). We are patching pyramidal neurons in neocortex, where the tau should be around 10-30 ms or more. The tau values based on on I-V tests in current clamp give us expected values around 30 ms. Can anybody explain why the tau values from membrane test (in VC) are so much off?
Thanks
When dealing with the fabrication of Ceramic membrane using Fly Ash & Clay or Kaolin, using sodium silicate as binding agent it breaks & turns into powder at room temperature after sintering at 800°C. Which binding agent is suitable to avoid the turning of membrane into powder form after sintering?
For unknown reasons I started to notice that after sometimes (0.5-1.0 hr) of operating crossflow filtration system, the flux increases steadily with corresponding decrease of rejection of the monitored ions (SO4 and Cl-). several types of membranes were tested with the same problem including NF270, BW30, NF, NFS, etc.
initially we thought something wrong with the cell so we changed it and used brand new Sterlitech cell. We also thought that some needle like pracipitates may have formed, we cleaned the whole system using 4 % cetric acid by circulation for 30 min then thoughly circulating UPW.
The cell we are using is Sepa CF Med/High Foulant System, 316 SS, 75 Mil with an active area of 140 cm2.
we are tried all different concentrations of polymer (Polysulfone) and solvents (NMP & DMF) to synthesis FO membrane. but unfortunately every time polymer penetration occurs across the fabric that results in poor water flux in FO testing.
kindly guide a way through which we can avoid penetration of solvent across the support fabric.
thank you
Hello,
I obtain western blot bands at desired region on the membrane but there is no space between the bands (attaching image for reference), may i know what could be the reason?
TIA
Hello, we have been struggling with the lots of background in our wb membranes probed with an anti-Streptavidin-HRP from Thermofisher (Pierce 21134). Samples contained biotinilated proteins. Every time there is some blobs somewhere and so much background that it is hard so see our biotinilated proteins. I attached the same pic with different contrast. Did anyone face the same problem?
All stepts have been performed with PBS 1X and here the protocol:
- After transfer, rinse off membrane for 5 min in PBS
- Block with BSA blocking buffer (1% filtered BSA and 0.2% Triton x-100 in PBS) for 30 min
- incubation with streptavidin antibody 1:2000 dilution ON at 4C
- Rinse off with PBS three times and do ABS blocking (10% adult bovin serum and 1% triton x-100 in PBS) for 5 min
- Rinse off with PBS three times and incubate with PBS for 5 min
- Develop with ECL for 5 min and acquire
I made 2 membrane samples of 91% CuO and 93% CuO using the sol-gel method. The SEM image of the grain is obtained evenly but the surface is slightly rough
📷
A similar ADC HKT288 that used a DM4 payload with anti-CDH6 monoclonal was discontinued over inflammatory and neurological adverse events. Would the adverse events be tied to targeting CDH6 or to payload toxicities?
I recently found an old bottle of opened chloroform (2.5L) in our lab, we would like to use it for RNA extraction, but not sure if it has been used for other experiments and would be exposed to RNase as there is no marking on it.
I have thought of filtering it but we only have 0.2um filter with PES membrane which is not compatible with chloroform.
I have also searched on the internet to find an answer but nothing appeared.
Has anyone experienced the same before?
please kindly help, thanks!
Hi guys. I was wondering if you guys have any suggestions on staining live cells for long time (ideally at least 5 hours)? I want to at least define where the cell boundary is, and hopefully it should be a red dye so it will have less crosstalk with my another green dye. I have tried several dyes but none of them satisfies me:
- Plasma membrane dye like CellMask and DiD: these dyes can visualize membrane well but just cannot retain too long (up to 1 hour for DiD and 4 hours for cellmask). They will be internalized into cells and can barely be seen on the membrane.
- SYTO 61 and 62. These dyes are for staining nucleic acid, but are actually good for seeing membrane cause it will also stain cytoplasm. The bad thing is that they make my cells super bright on green fluorescence due to unknown reason (probably cell stress) and just cannot be used for my purpose.
- SiR actin for staining the F-actin of cells. These are great in background and won't be internalized fast like PM dyes. However, my cells are probably not fully covered by F-actin and there are always space that is not stained by SiR actin but clearly is a part of cells.
I am running out of ideas now. The only option left is expressing fluorescent proteins tagged to other membrane proteins. CellBrite steady looks like another great choice since it is expected to retain signal at the PM for more than 24 hours. It might work but just could take too much time. Would appreciate it if anyone have experience on this topic ;)
Hi all,
Thank you in advance.
I labelled my membrane receptor (a GPCR 41 kDa; approx 80 kDa with SNAP at N-terminus and nLuc at C-terminus) with SNAP-AlexaFluor-488 (surface/non-permeable) and SNAP-647-SiR (permeable to the membrane). Lysed cells, collected total protein (stored on ice), stored at -20 dC for a week. Ran 10 uL supernatant on mPAGE™ 4-12% Bis-Tris Precast Gel, 10x8 cm. Electrophoresed first at 60V for 6 min (for protein to enter the gel) and then at 200 V for 33 min at room temperature in MOPS running buffer. Post-electrophoresis washed gel with tap water three times for 5 minutes. Scanned on Amersham Typhoon gel scanner using filter Cy2 (488 nm), Cy5 (635 nm), and Cy3 (532 nm). I see no problem with the Cy2 channel, but with the other two channels the images are weird - the gel appears granular, with white patches.
Note: while setting up the tank (just before loading the samples and filling the running buffer) I think I first slightly overtightened to create a seal but stopped and loosened it.
Please find the attached images
Please let me know if you need more information from my end.
Thank you once again.
Hi, I performed western blotting for the 8 times. In two of them I used wet-transfer and the rest of them assayed with semi-dry transfer. I checked my buffers, systems, gels and everything. I used two different primary antibodies which one of them is surely working. In the first 5 assay, I observed signal from the antibody that we know it is working. after we took images, I stained the membrane and observed protein bands on it. but now I can not observe any signal neither of them. and there is any protein bands on membrane. what could go wrong?
How does membrane selectivity impact CH4 recovery and CO2 removal efficiency in biogas upgradation?
I observed GFP-KrasG12D localization in the nucleus by immunostaining, instead of at the membrane where it is typically found. Could this be an artefact, or has anyone else observed this as well?"
best
marianne
I'm looking for protocols/sources that provide information about how to maximize the isolation of membrane bound proteins and general protein yield of unstimulated primary CD4+ T-cells. I have used RIPA and other ThermoScientific specialized protein lysis products and I have not succeeded.
These cells are isolated from fresh PBMCs.
My ultimate goal is to use Western Blot and ELISA as my protein assays.
Any suggestion is welcome. Thanks!
What is the optimal membrane material and configuration for efficient CO2 removal in biogas degradation? About membrane degradation units
What are the common fouling mechanisms in membrane biogas upgradation, and how can they be mitigated? Let us collectively list these down
How do operating conditions like pressure, temperature, and flow rate impact membrane performance in biogas purification?
I am getting extra peaks in my XRD graph of essential oil mediated zno nanoparticle which supposed to be of zinc hydroxy nitrates from the literature review. Would this sharp zinc hydroxy nitrate peak has any impact on the membrane stabilizing activity and antimicrobial activity.
Hello!
I am preparing liposomes using a membrane extruder.
The procedure is well-known and descibed extensively in the literature:
1. I start with a solution of the lipid in chloroform, then evaporate the chloroform. At this stage I know the mass of the lipid M.
2. I add buffer (volume V) and make several freeze-thaw cycles with vortexing.
3. The resulting mixture goes to extruder and becomes transparent upon several extrusion cycles.
What it the resulting concentration of the lipid? It should be lower than M/V due to adsorption of the lipid onto the membrane and the inner surface if the glass syringes. But what is the typical concentraion loss? Is it 10-15% of M/V?
What kind of material can I use as a membrane?
How does Membrane Scrubbing Technology remove impurities like CO2, H2S, and H2O from raw biogas to produce high-quality BioCNG (>97% CH4) while minimizing methane slip?
After extraction and separation of humic acid and fulvic acid, I need to purify them before characterization. What should be the size of dialysis membranes?
I have overexpressed my protein of interest (a single pass transmembrane protein) in C41 (DE3) E.coli cells. Currently, I am trying to solubilise the protein from the membrane using detergents. I have used DDM, OG, CHAPSO, LDAO, and LMNG, but nothing works. Has anyone faced a similar issue? Could someone possibly suggest a solution?
I have primary breast cells embedded in the hydrogel. When stained with EpCAM without the hydrogel, the staining was correctly localized to the membrane as expected! However, after embedding in hydrogel and using the same protocol for immunofluorescent, we are now seeing nuclear staining. Have you encountered this issue? Any recommendations on how to resolve it?
Hello,
I am currently working with Malaria Pf/Pan nitrocellulose membranes coated for malaria detection. After drying the coated membranes in an Incubator (overnight at 37°C), I've noticed that they tend to release moisture with time approximately within 15 days. Additionally, the (silica) desiccant turns pink, indicating the presence of moisture. This is causing issues with the stability and performance of the membranes.
Has anyone encountered similar problems or have suggestions on how to resolve this issue? Specifically:
- Is there a more effective drying method or a different drying temperature that could prevent the release of moisture?
- Are there alternative storage conditions or desiccants that could help maintain the dryness of the membranes?
- Does presence of moisture also affects the migration of blood sample from gold conjugate pad?
Note:
- Silica is activated properly.
- Incubator in not placed in DH
Any insights or recommendations would be greatly appreciated.
Thank you!
I am trying to coat my micelles and PLGA based nanoparticles with cell membrane using an Avanti mini extruder according to literature . I use 200nm membrane but my particles end up to be 400nm And high PDI. Anyone has experience using extruder for this purpose?
Does it exist some special technique for cutting ceramic membranes for Cross-sectional FESEM or SEM images?
Is there an established protocol to measure the permeability of bacterial membranes?
Thank you
I am trying to scale up a transwell migration assay for PBMCs and I have preliminary data from 96 well plate format, that shows that my cells migrate towards my attractant through a 5 µm polycarbonate membrane.
No I am trying to establish the same in the 24 well format and am having issues with this. With the same chemoattractant concentration, I see less than 30% of my migratory effect in the 24 well format when compared to the 96 well plate effect I observed so frequently.
The TC-treated inserts I use in the 24 well plate also have 5 µm pores, however membranes are made from PET (polyethylene terephthalate).
Could it be that my cells stick to the PET membrane more and fail to migrate to the lower compartment? Are there any other ways the membrane material could impact migration of PBMCs in this setup?
we have a nafion 117 membrane and we want to know if it has the properties claimed by the sellers. what are the tests available ?
I recently got new secondary antibodies but didn't realize they needed to reconstituted prior to use (first time getting fresh ones myself) until after I had finished washing my membrane with TBS-t. How long can my membrane sit in TBS-t? There is 30 minutes between end of last wash and when the secondary antibodies will be ready to use. Should I place the membrane in TBS while I wait?
I want to make sure that the culture medium contains membrane vesicles.
I prepared drug loaded HPBCD inclusion complex in two different ways.
1. In the first case, both were mixed under stirring till solvent evaporated and dried
2. In another case, the mixture was filtered using 0.45 micrometer membrane filter and the filtrate was was lyophilized to get the particles.
3. In the other case, both the drug and HPBCD were mixed in closed container using incubator shaker and filtered using 0.45 micrometer membrane filter and the filtrate was was lyophilized to get the particles.
It was observed that the loading efficiency was least in 3 followed by 2 (a little higher than 3) & then 1 (drastically high). Please help me with the possible reasons.
I have this problem with my western blot for the ABCG2 antibody on nitrocellulose membrane. Help please
I am performing in vitro drug release studies using franz diffusion cells. my drug is soluble in the release medium (3mg/mL) and for the duration of my studies. I have used 1 mL of 1 mg/mL of drug in aqueous solution in donor compartment and is also stable in it for the duration of studies. I have tried different membranes, and it doesnot seem to be problem either.. but still the cumulative release at 24 hours is not 100%.. i have also filtered my release media before use.. what could be the issue? I guess, I do not need to use any solubilizers as the release media volume is 12 mL and should be enough to dissolve 100% of drug from donor compartment if released
Several times by placing a restriction on the upper part of the membrane with the aim of preventing the drug from being added from this side, we tried to keep the drug at the top of the leaf, but the drug was added again from this side. Thank you for your advice.
I have prepared curcumin-loaded biopolymer nanoparticles and wanted to know how to check the drug release from the nanoparticles. The literature suggests nylon pouches, dialysis membrane, centrifugal ultrafiltration membrane, etc. Please let me know the best one for my work and the specifications to buy the appropriate one as soon as possible.
I am trying to insert cholesterol inside the DPPC membrane by using gmx insert_molecules -replace. But number of DPPC molecules replaced is much higher than the number of cholesterol inserted? Please suggest me a way to build the system by replacing desired number of DPPC.
Hello, I've just attempted a wet transfer of a western, and had set up the cassette and tank, and run @ 100V and set it to 90 mins. I had pre-chilled the transfer buffer - but didn't put the chamber in an ice bucket/add ice-packs (this is my first time attempting a wet transfer, I'm used to using semi-dry). When I walked in after 30 mins, the tank was genuinely steaming and boiling to touch. I switched it off and took out the gel/membrane, and am now blocking the membrane in some vain hope that there's something on it (but doubt it with the overheating and the fact it was only transferring for 30 mins). Has anyone had any experience of this - and it worked? And is there anything else you'd suggest other than setting the chamber up in an ice bucket?
Hi, I am preparing a porous Carboxymethyl cellulose membrane by adding DMF as non solvant. After addition of DMF into CMC, I stirred it at 70 degree for 4 hours and then put into glass petri dish to dry in a oven at 80 degrees. When all the water evaporates, then it starts to break. I cannot get a membrane. Also membrane is very fragile, it breaks just by touching it. Can you please let me know how I can resolve this issue?
I am working on an acetyltransferase that is highly unstable. Its pI is 6.65, and its molecular weight is around 18 kDa. The protein elutes at 1M imidazole and begins to precipitate immediately after elution. After testing various pH levels and salt concentrations, I have been able to stabilize it in MES buffer at pH 5.5 with 1M salt and 5% glycerol, by immediately diluting it after collection. The highest concentration I achieved was approximately 6.67 mg/mL, which was only possible by adding 50 mM EDTA post-elution. This addition seemed to stabilize the protein, but I am uncertain if this approach is optimal, and replicating the results has been challenging.
However, when I try to concentrate it using a concentrator, its concentration rapidly decreases after buffer exchange. I have tested the flow-through, and the protein is not present there. I have also tried flushing the concentrator membrane with buffer, but there is no protein stuck to the membrane either. Only a negligible amount is precipitated. I am unable to determine what is happening to the protein. My eventual goal is to crystallize the protein.
Thank you.
Hello everyone!
I am working on PVDF-based membranes for dye rejection and have encountered a recurring problem: after each rejection cycle, the membrane flux increases rather than decreases, although the membrane's separation efficiency decreased from 99.8% to 91% up to the 14th cycle. What could be causing this, and how can it be addressed?
I am looking forward to help from experts in the relevant field regarding this problem!
I am working with snake venom, specifically targeting toxins around the 15 kDa range. I need to remove higher molecular weight toxins (above 20 kDa). Would it be advisable to use a 20 kDa MWCO membrane to separate proteins above 20 kDa and then use a 10 kDa MWCO filter to concentrate the lower molecular weight toxins around 15 kDa which is of my interest?
Hi all,
Can I please ask, how can I calculate the pure water flux without knowing the surface area? I am using the NF membrane and I have the cross-sectional area but don't know the surface area.
I want to know the effect of TMP on the permeate flux.
Thanks
Hi,
I have a question regarding western blot. I performed total protein statin after trasfering membrane. The result was totally fine before (Figure 1). However, there were strange bubble-like spots over the membrane every time since last month (Figure 2). I have tried more than 10 times and switched the gel, PVDF membrane, buffers and the results were the same. I transferred the membrane under 200mA for 3 hours. Every one in our lab use the same transfer condition and share the tank for transfer. Their results were totally normal. I was wondering what is the problem?
Is PAMPA an effective option? what membranes can be used to evaluate the same? are there options available in cell culture using SHSY5Y cell lines?
Any comments are appreciated!
Thank you.
i was preparing gelmA by already reported method. but im unabke to seperate side product methacrylic acid and unreacted methacrylic anhydride. we donot have dialysing membrane facility nor we have lyophilization technique. kindly tell there alternatives
I have 2 proteins-one is 19KD and other is 5KD. I tried detecting both of them through western blot using 20% SDS gel. I transferred them into 0.45 PDVF membrane for 100mins, I could detect the 19KD protein but I couldn't get the 5KD protein.
Any suggestions will be appreciated.
Thanks in advance
Hi all!
I would like to know if there is a method to polymerize low MW cellulose triacetate (e.g. re-engaging in acetylation). Or this is not possible and the MW of my cellulose source should be high?
Thanks.
I am having an issue with the western blot transfer from last week, I see imprints of cassette on my membrane.
I am making a 14% gel and I run transfer for 2hr at 100V, after the transfer I see imprints of cassettes on the membrane.
I thought it could be because of the cassette was too tight or because of the temperature. So I keep in check for the cassette and temperature (1hr 45min) and repeated the western I see the same problem.
I used fresh buffers both the times.
Any suggestions how can I improve?
Problem 1: In my research, I encountered an issue with my wet transblot procedure: despite using 8-10ug of RNA sample and applying a voltage of 10 v for 2 hours in TBE transfer buffer, I observed incomplete transfer of the top band from the UREA gel (8M) to the nylon membrane upon examination.
Problem 2: for northern blot, I conducted prehybridization at temperatures ranging from 55°C, 60 °C, and 65°C, followed by membrane washing with SSC buffer and blocking with blocking solution. Subsequently, I proceeded wash the membrane in wash buffer and soak in detection buffer and applied CDP-Star on top of the membrane. The entire membrane exhibited fluorescence (is it normal?), later the resulting X-ray film exposure did not reveal the desired bands. Background noise quite bad. I would appreciate any professional guidance or suggestions to address this discrepancy."
I would like to run an invitro drug release experiment for chitosan nanoparticles (600nm) loaded with herbal extract using dialysis membrane method. However since its not easy to ascertain the molecular weight of the herbal extract because its mixture of different compounds. therefore I am not sure which is the best MWCO for dialysis membrane that I can use. I used 5Kda but nothing went through in 24hrs.
NF membranes sit between Reverse Osmosis (RO) and Ultrafiltration (UF) in terms of pore size. This allows them to remove a wider range of contaminants than UF but not as much as RO. However, current NF membranes aren't perfect at selectively removing certain contaminants while allowing desirable minerals to pass through. In optimizing NF membrane selectivity, could machine learning algorithms be used to design or predict ideal pore structures or surface functionalities for NF membranes?
For treatment of water (river), what is the life of ceramic membrane? For example, 5 years or 50 years? MF or UF?
Operation of ceramic membrane after usage: should be kept under water like polymeric membranes?
Aslamo alikom/ Greetings everyone,
I'm conducting a western blot experiment (8% SDS gel) and I wanna test 2 proteins, one is 95 KDa, and another is 35KDa. The antibodies I've for both are mouse Abs used at 1:1000 dilution and I use secondary HRP-conjugated at 1:2000 dilutions.
Ideally, I use to test them sequentially, but I'm wondering if it's possible to add the 2 antibodies to the incubation buffer (BSA/milk) and test for both in one go?
I would highly appreciate an answer for this. Also, if someone has done it before, I would appreciate the feedback/tips if any.
Thank you
Method of fixing exosomes on a slide, assuming that the membrane is negatively charged, the positive slide attracts exosomes
I want to study CO2 capture simulation on activated carbin filters in a duct how can I find the amount of CO2 adsorb by membrane?
I'm not sure how to interpret or fix the sample so that it creates a normal band. I used Percoll Gradient with density gradients to generate the questionable band. It's supposed to represent basolateral membrane fraction, and most people mentioned that the sample preparation is not good enough, but I'm not sure how I can make it "good enough".
I prepared a membrane for use as a dielectric material using the electrospinning method. their index is as follows: one shows 0.9 under 9 GHz and another 0.83 under 8.5 GHz but one shows 1.03 and another 1.09 under 10 GHz. After that, doubting the result, I re-examined and the same result is repeated. So can anyone give me any advice on this matter?
I am establishing a polarized gastric-epithelial monolayer culture on transwell system for bacterial infection studies. I use NCI-N87 cells and I culture them by replacing medium on every alternative day for 21 days. Later, I confirmed the expression of ZO-1 on 100% methanol (-20C) fixed cells. However, I face the following issues during this process.
1. How to avoid membrane curling while I cut off the membrane from the insert to mount on glass slide?
2. When I used 4% formaldehyde as a fixative to stain cell surface proteins, I found few cells or small cell clusters lying over the tight monolayer.
3. Is it necessary to use 21 days grown cells for bacterial infection studies? Because I see many highly cited papers also have used lesser days grown cells.
How to overcome these technicalities?
Any help is highly appreciated.
Thank you in advance.