Pellets - Science topic

The sample was made using flash nanoprecipitation, but it gave me a suspension in water, and it has been sitting on my bench for a month, and it has not precipitated on its own. I am trying centrifugation, which did not result in a pellet. So, I am wondering if the nanoparticles are just too small to settle.

I have made two pellets of apatite, and wanted to measure the vicker hardness data. But the issue is i am unable to see the indent mark. Is this because that the pellet is white and the optical microscope is unable to reflect it rather it is scattering the incoming light. What should I do in such cases. the magnification was even 400X which is last option available. Also I placed the pellet in the optical microscope at 1000X but still not image of the pellet surface.

The pellet is very sticky and will not go into solution. Can I vortex it? I need to add ~1ml of media to a pellet made from 10ml of lentil-X to get it to go moderately into suspension.

I am facing some trouble while pelleting tissue debris. I am using the attached protocol. After centrifugation and removing supernatant, I store my supernatant in -80c until bca estimation which is usually taking place in a day or two. I have observed that when I thaw my supernatant on ice and centrifuge again before bca, I get a pellet (again). This time it is little bigger than the pellet which I got after my first centrifugation. Is the speed, time of centrifugation or some other factor contributing to this observation.

His-Trple A protein construct into DE3 cell, pellet were suspended in 10mM HEPES ph 7.4 250mMkcl and 10%glycerol, The resulting pellet resuspending in 100mM Tris ph7.0,0.5M=mM EDTA,5mMDTT,2 M urea, TritonX-100,

Then treated with extraction buffer (10mM HEPES,250mMkcl,6M guanidine HCl but after chromatography (NI-NTA affinity)and dialysis protein aggregated, what should i do

His-Trple A protein construct into DE3 cell, pellet were suspended in 10mM HEPES ph 7.4 250mMkcl and 10%glycerol, The resulting pellet resuspending in 100mM Tris ph7.0,0.5M=mM EDTA,5mMDTT,2 M urea, TritonX-100,

Then treated with extraction buffer (10mM HEPES,250mMkcl,6M guanidine HCl but after chromatography (NI-NTA affinity) protein aggregated, what should i do

Dear all,

Currently I am working with the hCMEC/d3 (human cerebral microvasculature endothelial cells): http://www.emdmillipore.com/US/en/product/Blood-Brain-Barrier-hCMEC/D3-Cell-Line,MM_NF-SCC066

First of all, I need to establish the culture that was cryopreserved as I started to work at the new lab. There are few protocols available, incl. the data sheets from the manufacturer. I am following all steps:

-flask coating (with rat tail collagen type 1, 5ml of 50ug/ml for the t25 flask, before I tried 3ml) 1h incubation at 37 degrees Centigrade

-EndoGro-MV medium, all pre-heated and ready to go

-Rapid thawing of the cryovial (1ml pellet) and addition of 9ml of the medium followed by centrifugation 300g for 3min

Seems quite straightforward, though cell do not seem happy at all. I mostly see dead cells and it seems that there might be problems with attachment.

I already ordered the new cells and new collagen.

Nevertheless, if anyone is skilled with this cell strain and knows common mistakes or any tiny details worth consideration I will highly appreciate your help!

Best regards

1. Firstly, I am not getting any pellet even after following manufacturer's instructions.

2. I lysed it with 8M urea and 2% SDS and got 30ug/ul protein after estimation with bradford assay. Is this correct?

3. I am not getting any band when I run it on 12% SDS page.

Can anyone help me with the techique or assure me if I am going in a correct way or not.

Kindly suggest

Thanks

We found that we have this kit from Qiagen but labelled for serum/plasma samples. Can I isolate RNA from frozen cells pellet of MCF7 and MDA-MB231 cell lines using an RNA isolation kit intended for serum/plasma use?

Hi,

I have a question regarding DNA extractions.

I follow a magnetic bead based protocol for DNA extraction and at the end of the protocol, there is a step to spin down the DNA samples which are already in the elution buffer at 14,000 rpm for 10 minutes.

The rationale behind this step is to try and pellet the left over beads that carry over so that it doesn't affect the spectrophotometry when trying to quantify the DNA (A260/A280, conc. etc). And when you do spin down the tubes, you can usually see the tiny brown dot of beads.

My question is, does this spinning down also pull down the DNA or does it have no effect as the DNA will be in free suspension as its in elution?

I have attempted to test this my self by making repeat nanodrop measurements from the top layer, middle and bottom layer of the eppenforfs and found no substantial difference in the concentration but curious to see if anyone has any experience/input regarding this?

Thank you in advance.

I also need to know why the beaks of layer birds are trimmed, but not those of broilers.

Hello everyone! Currently, I am preparing RNA samples from a minimal material. I would like to use GlycoBlue to increase the yield and make it easier to work with the pellet. Do you know if the presence of the glycoblue could affect the downstream application, which includes mass spectrometry?

I'm getting dark blue colonies and lawns when I'm plating E. coli cells transformed with plasmid containing E2 Crimson as the gene of interest under high copy number origin of replication and constitutive promoter in both LB as well as M9 agar plates. Similarly after centrifugation of such cells, I get dark blue pellets of the live cells.

Can anyone explain why I'm getting such blue colonies/lawn/pellets? If anyone can direct me to any literature mentioning such observation will also be extremely helpful.

Dear Colleagues,

I want to coat YBCO(123) superconductor pellet for moisture protection.

The coating needs to be thermally conducting, electrically insulating.

Please suggest which coating can be used for this .

Thanks in Advance.

Nityananda Das

Looking for some advice!

We are isolating His-tagged proteins using NI-NTA beads (can describe the protocol if needed) and then concentrating the final product (6mL) with PEG down to about 1mL, rinsing all the PEG off, and then dialyzing against (A)PBS modified to be amphibian-compatible.

APBS (amphibian):

We dialyze the concentrated protein overnight (12+ hours) in 4L of APBS pH 7.4 at 4 degrees C with a stir bar slowly stirring the solution. The buffer that the protein was eluted in is 500mM imidazole, 500mM NaCl, 50mM NaH2PO4 and pH 8.

Today we realized that the protein was forming a pellet at the bottom of the eppendorf tube after it was taken out of dialysis.

We need to use this protein in vivo and are not sure how to resuspend it and keep it from pelleting out. Also looking for ways to prevent it from pelleting out.

Happy to provide any more context that would help answer questions.

Thanks!

Hello everyone

exosomes extracted by 8% PEG were analyzed by DLS but the result only show particles with less than 1nm in diameter even when during tbe extraction process pellets were visible. pellets were broken up by gentle pippeting, could this be the reason why the DLS result show those readings? any idead or suggestions for troubleshooting are greatly appreciated

thanks

Nima

I've pelleted down the overnight ampicillin supplemented cultures of DH5ALPHA for pET22b isolation 5 days ago and stored them on -20°C without adding any buffer or glycerol. Would the cells be viable for mannual plasmid isolation?

These peaks are pure KBr pellet. What could be the reason that KBr has given a peak? Has it absorbed moisture? Should KBr can be kept in the oven all the time or can it be kept in a desiccator?

Is it possible to remove the KBr peaks first with a pure KBr tablet as background?

i am trying to lysis the bacterial pellet (500ml culture pellet) with 50ml of lysis buffer ( 0.5 m Tris HCL, 5M NaCl pH 8.0, 100mM PMSF, Lyzozyme ) by sonication (Amp 20, Esp time 10 mins, Pulse on 15 SEC, Pulse off 10 sec ) but its not working. i have used the same method previously, it worked well. But facing problem for the first time. what might be the reason ? or any suggestions

We are recylcling rPVC with a proprietary formulation that, among other things, contains DOP and wax. The pellets immediately stick together when they are cut by the cutter and form a string (imagine the beads in a rosary.) This is problematic becauase the product should be in pellet form not string. How could we solve this problem?

Conventional carbon pellet fabrication relies on binders, but for my X-ray and transmission IR experiments under microwaves, I need binder-free pellets. Since these experiments involve heating, organic binders are not suitable. Is it feasible to create thin, mechanically stable carbon pellets for X-ray/IR applications using binder-free methods with various carbon sources like activated carbon or carbon nanotubes (CNTs)? Is there anyone with experience or ideas for creating these binder-free carbon pellets?

I have applied a general protocol. In short:

20uL of serum were added to 8 volumes of a cold TCA+ Acetone solution. After a light stirring I incubated for 90 minutes at -20 followed by centrifuge at 15,000 rpm for 20 minutes. The pellet was washed with acetone and then lyophilized.

The coloration revealed that the albumin was not completely removed from the serum, and the pellet did not resuspend properly. I tried resuspending the pellet in Laemmli Sample buffer.

Can anyone help me?

I am working on protein biopharmaceuticals, we have engineered a novel antibodies (4 variants) and take a service from the company to procure our gene of interest in pPIC9k vector. Before confirming the vector we have verified everything regarding our construct and it was all good according to us. After receiving the genes, I proceeded to make its copies and transformed it in to the E. coli DH5a, it was successful as we cross check it with the double digestion and dropout bands were at expected size. Then I further proceeded for P. pastoris transformation. Following steps I have done;

1. Competent cell were prepared with Electroporation Transformation protocol which is given in this article. ( )

2. Then, I linearized the plasmids with two enzymes as we opted for two restrictions site. ( Variant 1 & 2 have SacI site and Variant 3 & 4 have BglII site)

3. Electroporation was done by using Eporator (Eppendorf Electroporator) at 1700 V. In this we do not have option to set other parameters.

4. Immediately after the electric shock I added 1M sorbitol then incubate it for 2 hours with no shaking then incubate it for recovery.

5. After this I pellet down the cells and thrown the 400 ul sorbitol and again resuspend the cells in remaining media. And then I plated this on MD plate (200 ul).

6. After incubating it at 30 Deg for 4-5 days, we were sometimes able to get colonies and sometimes no colonies were their.

7. The colonies which we get was false positive and were not able to express our desired protein. (Confirmed with SDS & WB)

I have tried multiple protocols (Condensed protocol, used G-Bioscience competent cell preparation kit) and change electroporators also. I also changed my P. Pastoris cells, tried with new cells also, I tried with GS115 cells also, tried with different DTT as well as its concentrations, I tried with different OD from 0.8 to 1.5.

The thing is that from 4 of the variants, I only able to express one variant which was at initial phase only. Also, we have some other molecules (proteins) which we have already expressed it from the same system with the same protocol. Now I am keeping them as my positive control. But now that too have shown the same problem.

Kindly if anyone have any clue regarding this please suggest me. Any small suggestion can make very huge contribution.

I have successfully done this before by adding PFA in small pellet form and agitating for around half an hour, then adding solid sodium hydroxide to clear the solution. However, I would like to try other forms of solid PFA like fine powder and cyrstalline forms and have not had the same results. I was wondering if there is anybody who has experience of dissolving solid PFA into acetone generally and if they could let me know a method that worked for them. Many thanks in advance.

If anyone has knowledge about my question, kindly help me

I am not able to get good literature and the physics behind how first these grains and grain boundaries arises out of no where when we make a pellet to study its dielectric properties and then how are they so important when studying its electrical properties.

Will love to hear its physics or provide me with good book which discuss these things and concept regarding the concept behind them!!

Hi, I culture HACAT mammalian immortalized cells. I am interested to check the protein changes by western blot and cell cycle analysis by flow cytometry. Can i store the harvested cell pellets in -80 freezer without any storing reagent before performing western or flow cytometry?

Thank you

I did this twice.

I couldn't see any pellets after adding isopropanol the 1st time

The second time, the yield and ratio was really bad.

Hello, I am currently having problems with RNA extraction.

I am using mouse liver (C57BL6J), and I have extracted RNA from mouse liver before.

Before this experiment, my final RNA pellets were white before drying, and became transparent after drying.

However, this time the pellets had yellow parts like the attached picture, and didn't easily become transparent. I dried the RNA pellet up to 40 minutes, and still the pellet was white.

Did anyone encounter the same problem as I did?

I am trying to prepare samples for IP. The pellet size is good and I am using 1% Tx-100 and using a syringe to further lyse the cells and maintain protein-protein interactions. I keep having a small amount of pellet remain in the solution and protein concentration ends up being too low for IP. No matter how much buffer I add or I pipette/syringe the mixture, the pellet does not disappear and I feel like I am losing protein concentration because of that. How can I increase my protein concentration?

DNA pellet in wash buffer seems different in my experience. Is this a DNA molecule or something else?

How to determine tannin content in fodder pellets?

I am cloning an overexpression plasmid with my protein of interest tagged with mScarlet. After transforming my ligated product into DH5α bacteria and plating on LB agar, I noticed colonies with a pink tint to them. Miniprep pellets and even DNA elutions were also pink. The plasmid has been sequence verified and has the confirmed sequence. After looking online, I wanted to be sure this is not yeast contamination so the USER cloning was repeated. Again I saw both white and pink colonies and had pink pellets upon centrifugation. This issue has persisted across multiple batches of agar plates. Others in the lab have been doing transformations and have not come across this so it seems to be specific to my plasmid only. Could this be expression of mScarlet from my plasmid? This would be weird it is under the expression of mammalian promoter and none of my other mScarlet plasmids have had this issue. I've attached some photos for reference (arrows pointing to pink and white colonies).

Hi all,

I have been scouring the internet for a protocol for defrozen PBMC fixation and embedding.

Currently I am defreezing PBMCs with an in-house protocol which gives me ~95% viability. After pelleting and washing (PBS no BSA) I fix my sample with 4% PFA overnight (~16h) followed by 2X wash with PBS (no BSA) and spin down @ 800g for 8min in 15mL Falcon tubes.

The issue I am encountering is massive cell loss and usually end up with barely 5% of my initial cell count.

Considering that I use standard histology protocols I have a hard time understanding what the issue is. Of note, cells maintain their morphology after fixation and clearly the loss intervenes at the washing stage.

Would using PBS with 0.2% BSA for the post-fixation help me pellet better and keep more cells?

Overall, if anyone has a good protocol to recommend for FFPE preparation of PBMCs I will gladly take your advice.

Thanks in advance :)

I am extracting proteins from plant samples and am currently attempting to use the TCA/Acetone extraction method. I am having trouble resolubilizing my sample after drying the pellet. I collected the pellet during the extraction process as per the protocol, but I am confused about whether the pellet is the actual plant residue from the initial grinding (using liquid nitrogen) or if it is the extracted protein, since the supernatant is discarded throughout the extraction. I have not yet proceeded with any treatment using DTT/IAA or protein digestion - I simply want to test the sample using a Bradford assay and intend to resolubilize it. If anyone able to answer my questions, it would be very helpful. Thank you

I extraction RNA from Physcomitrella patens (Moss) Following the protocol of RNAiso Plus. Firstly using a tissue lyser and added 1 ml RNAiso plus and vortex. next step Add the chloroform after centrifuge I separated the top liquid layer but the problem is yellow color liquid. I could not avoid the yellow color at ned of the extraction process after the precipitated pellet is brown.

unfortunately, i couldn't find any proper research papers about the influence of the flow-return temperature of the heating network (District heating) on the investment costs ( CAPEX ) of the heat generators such as CHP, Heat Pump and Biomass (Pellet,wood) boiler. I wanted to draw up a comparison between the heat generators in order to determine the most sensible variant for a given flow/return temperatures in terms of cost-effectiveness. I'm planning to establish a correlation between the temperatures and the capex.

I would really appreciate any help.

Best regards.

hello I start work in analyze lab with FT-IR instrument

I am sending the pictures of some of the samples that have been sent to the laboratory for analysis. I wanted to know if it is possible for you to guide me on how to prepare these samples?

Previously, we used to analyze samples by preparing KBr pellets, but it seems that these samples cannot be ground and mixed with KBr.

I've pelleted down the overnight ampicillin supplemented cultures of DH5ALPHA for pET22b isolation 5 days ago and stored them on -20°C without adding any buffer or glycerol. Would the cells be viable for mannual plasmid isolation?

I am using the hanging drop method to develop individual differentiated adipocyte spheroids from the stromal vascular fraction of adipose tissue. With our current timeline they end up 100-300um. My problem is, when I remove the spheroids from the matrigel droplet with ice cold 1x PBS, I pool multiple spheroids from the same treatment into one tube for RNA extraction, and the spheroids float and will not pellet when I centrifuge. They float, and I cannot remove the excess PBS without aspirating a spheroid. I’ve tried 300x g, 500x g, even 1000x g for 15 min RT. I don’t want the spheroids to lyse in the PBS, I want to remove the PBS so I can add lysis buffer. Too much residual PBS is what I think is contributing to my low RNA concentrations (1.1 - 6ng/ml for 20 pooled spheroids). I am using the micro RNeasy kit from qiagen, including the carrier RNA they provide for low RNA samples. This picture is after 1000x g centrifugation for 15 min. Any help or advice would be much appreciated!

We are experiencing different problems pelleting fibroblast:

a) From the resuspension of the comercial vials (after defrosting, we resuspended in 5ml complete DMEM + L-Glutamin + FBS + Streptomycin + Penicilin)

b) From a suspension of a 25mm2 flask at confluence (same medium).

In both cases we used 50ml falcon tubes and 25ºC.

- We don´t see any pellet by centrifuging at 1500rpm/254 rfc

- We see just a very tiny deposit when centrifuging at 1900/400 rfc. But we are afraid the cells could suffer.

Any recomendations? Thank you very much for all your help.

Im extracting RNA from tomato leaves by the TRIZOL method but since the chloroform phase separation I can see that the aqueus phase is brownish and the pellet is totally brown. When I evaluate the RNA 260/230 rate is lower than 1. I have tried different methods such as CTAB at 1.5% in buffer, double chloroform phase separation, etc.

How can I improve this result??

MgO NPs were synthesized by sol-gel method.

i got Raman peaks at 1022 and 1400

I'm trying to achieve bonding between two different substrates through thermocompression using indium as an adhesion layer. After depositing indium through e-beam evaporation, the thin film is white and not metallic like the pellets. Is this common or an issue?

We are facing hurdles in achieving desired deposition rates while depositing cathode-based materials via thermal evaporation. Despite starting with a vacuum setup reaching up to 10^-7 torrs, initial deposition stages show promising rates. However, these rates decline significantly over time, seemingly linked to the depletion of lithium content. Even with the power input increased to 200 W, I still fell short of reaching the minimum desired deposition rate (>0.02 nm/sec). Although my power supply can go up to 2000 W, going beyond 200 W is problematic due to significant power fluctuations with these cathode materials.

Since electron beam deposition isn't feasible due to challenges in pellet formation without binders, I'm considering the possibility of incorporating dopants or flux agents to lower the melting point. This could potentially counteract the observed reduction in deposition rate.

I'm seeking advice on optimizing thermal evaporation parameters or exploring alternative methods to enhance deposition efficiency for cathode materials, considering the limitations and basic principles of material deposition processes

Everytime I centrifuge my H6c7 cells, the cells don't pellet properly. Though there is a pellet formed, the supernatant has visible particles which I believe to be the cells. I usually centrifuge at 300 x g for 5 mins. I have tried increasing the centrifugation time to 10mins and also increasing the speed to 500 x g. Despite this, I always lose cells dramatically. Please has anyone had a similar problem with this cell line, or another cell line? If so what method did you try to solve the problem?

For WGS, we need to obtain pellets of Avibacterium paragallinarum (Pasteurellacea-like baceria). What are the preffered centrifuge conditions for this bacterial family? Thanks

I have done Impedance analysis of a pellet with silver pasted electrodes in Hioki3536 LCR meter, but my data is so messy and Zig zag, in nyquist plot. I have make the Pellet again an the data is showing the semicircular trend. Please explain to me the possible reason that can be for this, is my silver pasting is wrong, is there any reason from the Pellet formation, or any other possible reasons.

...

I'm unable to currently continue the work on my thesis due to losing a huge number of cells in wash steps (up to 75% after 2 washes).

I'm using 2 cell lines Kasumi-1 and Molm-14.

I'm working in 1.5ml microcentrifuge tubes and washing with 1x PBS. The cells are not being burst as I can see them if I save the supernatant and run it through the cytometer.

I was previously able to pellet the cells for washing after fixation, but in a swinging bucket centrifuge at a collaborator's lab which I do not have access to on a daily basis.

I have currently tried 200g for 3, 5 , 10, 15 and 20 minutes; 400g for 5, 10, 15, 20 minutes; 500g for 5 minutes; 800g for 5, 10, 15 , 20 minutes; 2000g for 5, 10 minutes. Some conditions provided slightly better or worse results, but none were good enough to move on with my full protocol, which requires pelleting and resuspending the cells ~10 times.

I have also tried adding 0.5% BSA, but it did not seem to help and disrupted the counting of cells in the cytometer.

This is my first project working with eukaryotic cells so I am a complete beginner, so any advice would be appreciated. I have deadlines approaching and am at my wits end at this point.

Hi all,

I am trying to use the 3devo filament maker to make my own filament.

1 I first dissolve the ABS pellets bought form 3Dxtech and then use ultrasonicator to mix the ABS and nano SiO2 particles.

2 And then I vac the solution and pour out to make a sheet. Then I use a paper shredder to cut it into pellets again.

3 However the pellets will always melt at the feeder and form a huge particl and it will stuck the feeder.

4 I think settings will be good, but I can't get some uniform pellets. Could you please help me?

Almost all of the protocols that I have located utilize sequential centrifugation to eliminate impurities such as cell organelles, extracellular matrix, and other compounds.

I am attempting to isolate plant exosomes and have incorporated low RPM's (5000) resulting in about 3700 x g and signification at 40 hz for 3 minutes. I am concerned that I am destroying the exosomes via sonication. If anyone can provide some guidance that would be great. I do not need the last step in these protocols as the resulting supernatant and exosomes will be used topically.

Thanks, Michael

Let us take the example of Zn with Fe3+ reaction. Zn wants to oxidize and Fe3+ wants to reduce to Fe2+ with that electron. Now assume there is no cell.

1) If i simply add Fe3+ solution to Zn pellets how would the reaction proceed?

2) Do I still need to consider the Nernst Plank eqn for transport of ions in this case since there is no external voltage applied?

3) What are the constitutive equations that I need to look at trying to model this cell?

Hello everyone,

I am supervising a master thesis student whose goal is to compare different lysis methods for recombinant protein purification. We are working with E. coli pellet and he is interested in doing osmotic shock for cell lysis. I have never done such method as I usually use a homogenizer and lysozyme.

Has anyone ever done this method? How effective is the lysis?

I am open for inputs for the optimized methods that work for you!

The supernatant will be loaded and purified using IMAC.

Thank you in advance!

Best regards,

Lieke Widowati

I'm currently conducting myeloma cell (MM1s) culture for experimentation. I'm using RPMI-1640 medium with 10% FBS and 10% penicillin+streptomycin. I seed 5*10^6 cells in a 150mm petri dish with 20ml of medium and maintain them every three days. However, with each culture, the cell number doesn't increase significantly, and viability fluctuates between 40-60%. After just a couple of maintenance cycles, all cells die. I need a large quantity of cells for my experiments, so I'm trying to scale up the culture while maintaining and conducting experiments simultaneously. However, I'm stuck because I can't perform experiments, and all the cells are dying. What could be the problem? Here's my maintenance protocol:

Even with this method, both viability and cell numbers are very low. I reduced the centrifuge speed because lower rpm CFG was said to aid in dead cell separation. Previously, I centrifuged at 1800rpm for 3 minutes, but when I changed to lower rpm, viability increased by about 20% (approximately 60%). The pellet appears visible. When I observe the cells under a microscope during culture, they don't seem to be in good condition. Changing the media doesn't yield different results. What could be the problem?

I began by processing a 500 mg plant sample, grinding it in 5 ml of phosphate buffer. Following this, I centrifuged the mixture for 15 minutes and collected 1 ml of the resulting supernatant. To precipitate the proteins, I added 1 ml of TCA to the 1 ml of supernatant and left it overnight. After centrifuging the sample at 3000 rpm, I discarded the supernatant and retained the pellet. The pellet was then dissolved in 2ml NaOH (0.01 N). From this solution, I took 0.5 ml and added 0.5 ml of water along with 2 ml of Bradford reagent. Finally, I measured the absorbance at 595 nm. The reading for the sample was determined to be 169.82 micrograms/ml.

My question pertains to how to estimate the total protein content in the 500 mg sample, allowing for the determination of the protein percentage within my sample.

1. Scrape cells and collect by centrifugation (500 × g, 5 min, 4 °C)

2. Resuspend cell pellets in lysis buffer and incubate on ice for 10 minutes

3. Centrifuge at 500 x g for 5 min at 4C

4. Resuspend pellet in lysis buffer one drop at a time

5. Incubate on ice for 10 minutes

6. Add 2.5 volumes of sucrose buffer by pipetting slowly into wall of tube

7. Centrifuge mixture at 3500 x g, 10 min, 4C

8. Collect supernatant as cytoplasmic fraction and proceed to protein extraction

9. Keep nuclei pellets and proceed to protein extraction

1. Wash cell fractions using ice cold PBS

2. Centrifuge at 500 x g for 5 minutes and aspirate PBS

3. Add 1 mL 1XRIPA buffer (with protease inhibitor) for 1x10^7 cells

4. Agitate the contents in microcentrifuge tubes for 30 minutes at 4C

5. Centrifuge tubes at 12,000-16,000 x g for 20 minutes at 4C. Collect supernatant in fresh tubes and place on ice. Discard pellets.

6. Perform BCA assay to determine protein concentration

I am working with a DNA binding protein 180 Kda. after Heparin Purification or Gel filtration, when i used to centrifuge the purified protein at 10,000 xg, 10 mint, pellet is observed sometimes and after pellet formation, the concentration of purified protein is reduced significantly. Centrifugation is important for homogenizing for downstream assays. So how could i determine that which centrifugation speed is suitable to homogenize this protein??

Good morning,

I used TCA/aceton protocol to precipitate proteins. I incubated vesicles from yeast with TCA. After centrifugation 14000xg, 10min at 4°C I saw black pellets in each samples... What could happen? Is there any option to purification such protein pellets?

Currently I am trying abcam protocol for it.i checked the cell lysis by trypan blue stain which is more than 90 %. but I am facing following issues

1. unable to remove beta actin in mitochondrial fraction.

2. I am able to see mitochondria pellet (cell-1-1.5 X 10^7 cells) but the protein yield is very low 30 ug. and band of interest-SDHA is also very less on western blot compared to whole cell lysate when same amount of total protein (30 ug)

We recently got a new cell line in the laboratory but are having difficultly amplifying the resulting gDNA in any PCRs. We use the standard Bioline isolate II genomic extraction kit which we haven't had any trouble with for other cell lines. When DNA extracted from this cell line we get a workable yield (25ng/ul-180ng/ul depending on # cells used) and the 260/280 + 260/230 are always correct.

We have tried amplifying this DNA in >6 different primer sets which are all well optimised and consistently work for other cell lines yet it will not work for this. For reference we have completed this gDNA extraction with fresh cells on 5 separate occasions alongside other samples to rule out user error and all other samples have worked except for this one.

We have tried:

- Reprecipitating the DNA using a standard salt-ethanol protocol

- Using primer sets for a different gene

- Reducing DNA to 5ng, 10ng and 20ng (we use 50ng for standard 25ul reaction using ThermoFisher Phusion II enzyme) to dilute any PCR inhibitor

- Increasing DNA to 100ng, 200ng

- Reamplifying template (this gave non-specific bands)

- Washing cell pellet in PBS before extraction

- Extra washes during extraction process

It appears there is some cell-specific contaminant preventing PCR amplification.

If anyone has seen this before or knows of any troubleshooting recommendations that would be great!

I am currently undertaking the synthesis of AgNP utilizing plant extract as a bioreductor. The synthesis procedure involved reacting the plant extract with 1 mM AgNO3 while optimizing the ratio. Various ratios of AgNO₃ to plant extract were explored, including 5:5, 6:4, 7:3, 8:2, and 9:1. Subsequently, UV-Vis characterization was performed to identify the ratio that yields a single peak in the range of 380-420 nm with the highest absorbance. Upon determining the optimal ratio, the synthesis was scaled up to a total volume of 250 mL. However, post-centrifugation at room temperature, only a minimal pellet was obtained, with the colloidal solution predominantly adhering to the bottom of a small 15 mL tube.

I express my protein in E. coli. After pelleting down the induced medium, I begin lysing the cell pellets through sonication. Suddenly, I am observing that my cells are not lysing. What could be the reasons? How can I fix this?

Hi everyone,

I have never worked with Inclusion Bodies so do not have a method that really works.

My protein is recombinant ß-casein that is produced both in the soluble fractions of the pellet and the insoluble ones. As for the pellet, we did resolubilization with buffer containing 8 M urea and directly load it on the HisTrap columns. The flow rate was 0.5 mL/min (the slowest on ÄKTA Start machine) but still overpressure often happened. I was pretty sure this was not the best method to purify the desired protein from the resolubilized pellet, and some kind of intermerdiate method needs to be done before purifying on the ÄKTA.

Therefore I need advice of either:

1. What I can do after resolubilization to alleviate the pressure before loading the sample to the HisTrap column? (my protein has a His Tag)

or

2. Is there any other method that can be used to purify the ß-casein? Maybe precipitation?

Column chromatography is not a must so other methods are welcome.

This is a trial and error for me so I can try any suggestions.

All suggestions and discussions are welcome.

Thank you in advance.

Hello all,

I present here a problem to see if someone has encountered it and can provide some clarity. We are trying to isolate neutrophils from patient blood in tubes EDTA. We are following the protocol in

which is quite straightforward. We have a lot of experience using Percoll and Ficoll solutions, so I am positive it is not a matter of bad layering, wrong calculations when preparing the solution, wrong centrifuge settings (acc-brake, etc.)

According to the protocol, there are 2 centrifugations, which should result in a separation such as:

-Serum

-Lymphos

-Percoll

-Neutros

-Percoll

-RBC

After the first centrifugation (200g), the blood trespassed the 62% layer, staying in the middle of the tube. However, after the second centrifugation (400g), nothing happened, the blood was unable to trespass the second layer. Moreover, we started to increase the speed of these centrifugations and went up to 1000g (in for a penny...), and still nothing happened. The blood was unable to trespass the second layer, and I am quite puzzled, since 1000g is a very high force and you would expect most cell types to pellet. See attached.

There was a fine cloud on top of the blood and after going to the cytometer we could see all immune cell types, meaning no immune population was isolated.

What could have gone wrong? I would understand bad separation, low yield, etc. But a whole 15ml of blood not depositing, even a 1000g?

Thank you in advance.

we are facing pellet carking in the DRI pellet with >4% TiO2 in the raw material. \is there any solution to control the pellet cracking in the downstream process. |Thanks in advance

I am facing an issue regarding the concentration and purity of RNA, the 260/280 ratio shows contamination.

1. Aspirate media from plated cells.

2. Rinse cell monolayer with room temp PBS only once.

3. Scrape the cell monolayer with cell scraper.

4. Transfer cell to 1.5 ml microfuge tube. Centrifuge at 5000 rpm for 5 min.

5. Remove the supernatant and added 400µl TRIzol. Mix till the pellet dissolve by pipetting thoroughly.

The volume of TRIzol added should be as follows:

For 1 well of a 6 well plate or a 35 mm plate use 400 µl TRIzol.

(After this The lysate is stored at -80 degrees C).

6. Before the phase separation the lysates are thawed on ice.

7. Incubate the lysate for 5 minutes at room temperature.

8. Phase separation: Add 0.2 ml of chloroform to the supernatant (per 1 ml of TRIzol reagent).

9. Shake vigorously for 15 seconds and incubate at room temp for 2-3 min.

10. Centrifuge for 15 min at 12,000 x g at 4ºC.

11. Following centrifugation, the mixture separates into lower red, phenol chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube.

12. RNA precipitation: Precipitate the RNA from the aqueous phase by mixing with ice cold isopropyl alcohol. Use 0.5 ml of ice cold isopropyl alcohol (per 1 ml of TRIzol reagent used). Incubate for 10 min on ice (if cloudy, precipitate for an additional 10–15 min).

13. Centrifuge at no more than 12,000 x g for 15 minutes at 4 degree C.

14. After centrifugation, a small white RNA precipitate should be visible on side of the tube at this point.

15. RNA wash: Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol (per 1 ml of TRIzol reagent used). Mix by flicking and inverting tube.

16. Centrifuge at no more than 7,500 x g for 5 minutes at 4 degree C.

17. Repeat the above washing procedure once. Remove all leftover ethanol.

18. Air-dry RNA pellet for 5-10 minutes.

19. Resuspend the pellet in 20ul RNase free water.

20. incubate at 55ºC for 3 min.

21. stored at -20 degree C.

The nanodrop reading is very low (in tens and hundreds) and the 260/280 ratio is coming in 1.8-1.9 range. What is the issue I am not able to understand. Is it due to a handling error or do I need to change in protocol something?

Hello, I am using the conrad chromatin-associated RNA extraction protocol.

Everything was fine, until it was time to resuspend the pellet corresponding to the chromatin fraction, since a kind of jelly was formed, which absorbed the water.

I need to know how to solve that and if it is normal.

How can I solubilize the pellet?

I'm trying to isolate Lysosome using a percoll (sigma, cat# 4937) gradient of 57.6%, 43.2%, 28.8% and 20.2% (Ref: Lysosomal and non-lysosomal peptidyl hydrolases of the bloodstream forms of Trypanosoma brucei brucei; John D. LONSDALE-ECCLES and Dennis J. GRAB). I'm centrifuging the gradient at 25000 rpm for 35mins in a swing bucket rotor as mentioned in the paper. After the centrifugation I am taking 2ml of each fractions and diluting it 5X with the homogenization buffer and re-centrifuging it again at 20000rpm for 30mins. The problem is After centrifugation I am observing a percoll pellet along with the protein pellet. Because of the percoll pellet I'm facing problem in sample preparation for western blot. Please suggest me on what should I do so that I can get the lysosome enriched protein pellet from the fractions without pelleting the percoll.

A soluble protein is coming in pellet