98 Questions 62 Answers 0 Followers
Questions related from Jeff Zaki
I am trying to identify bacteria on skin that can utilise lactic acid as carbon source. So i intend to swab skin and then grow bacteria on minimal media supplemented with lactic acid. My question...
12 December 2018 3,525 0 View
Hi, I am hoping to get opinions/input since I am undergrad and I see certain trends that I am highly curious about. I have been doing lab rotation to get an extensive research experience....
10 October 2018 9,859 0 View
I applied some mechanical stresses on cells and discovered that FAK and p38 MAPK are activated by phosphorylation. For the purpose of publishing, i feel that it will be great if i can look for...
04 April 2018 5,888 0 View
hi i need advise on the matter i performed RNA-seq after exposing cells to mechanical stress. I had designed a device to deliver this stress to cells. Looking at the RNA-seq data, i see some...
03 March 2018 5,403 8 View
Im looking to expose cells to hypoxic conditions in minutes (
03 March 2018 5,658 0 View
How is proliferation assessed by manual cell counting? I will be happy if anyone can provide protocol on this. I have been told this method is much useful than indirect assays like CFSE but i...
12 December 2017 2,615 0 View
why when wanting to look at genes that respond at say 1hr (early response), 6hr(intermediate response), 16hr (late response) it is better to look at RNA expression rather than protein? has it got...
08 August 2017 4,701 2 View
hi i am not sure if it is alright to analyse my data this way. So i prepared 3 samples of controls, 3 samples of condition at 1hr exposure, 3 samples of condition at 6hr of exposure. i collected...
08 August 2017 7,852 5 View
Let say if i stimulate a cell at 1 hr and 6hr, the RNA expression upregulated at 1 hr may not be reflected at 6hr, but same protein upregulated at 1 hr will be reflected at 6hr. How do i explain...
07 July 2017 876 0 View
why is it that people usually look at mrna expression rather than protein expression when conducting a time course gene expression analysis?
07 July 2017 9,473 0 View
i intend to apply mechanical pressure on cell to look at mechanotransduction pathways. i will be using a SILAC based phosphoproteomics. Can the data also indicate that the cell is undergoing...
06 June 2017 9,061 0 View
Hi i would like to know because i did not wash off the ponceau stain from the nitrocellulose membrane thoroughly, and as a result the recycled primary antibody solution has ponceau dye in them....
05 May 2017 9,146 4 View
hi i am really confused here. so let me describe what my experiments is all about. I want to look at whether certain signal pathways are activated upon mechanical stress onto cells, so i exposed...
05 May 2017 6,659 2 View
Are the cells undergoing apoptosis or is this just a mere direct effect of putting physical mechanical strain on the cells?
04 April 2017 7,231 0 View
are any of these antibodies good indicators for viewing plasma membrane structure? - cdc42 cd9 cd81 integrin
04 April 2017 9,357 3 View
hi cell signalling company recommend that i use 1:1000 dilution for phospho-Akt but at my disposal is only about 1ul of primary antibody left. i usually use a falcon tube at 4ml total primary...
03 March 2017 7,891 0 View
hi this is the first time im detecting phosphoproteins. This is the recipe for my lysis buffer: 250ul of 1M hepes 600ul of 5M NaCl 10ul of 1M MgCl2 20ul of 0.5M EGTA 200ul of 1M B-glycerol...
03 March 2017 295 3 View
Hi I constructed a cell compressions device and I wanted to show that my device is not adding extra variables like hypoxia so I did western blot detection of hif1a and it came negative which is...
03 March 2017 8,531 1 View
I have only been performing normal defection of total proteins and is wondering what changes I should make for detecting phosphoproteins? 1) do I wash cells with 1x tbs instead of pbs before...
03 March 2017 9,264 6 View
i did nuclear lamin staining on transwell insert which contain monolayer of MDA-MB-231 cells that were mechanically compressed. do you see any nuclear abnormalities? red: actin, green: nuclear...
02 February 2017 6,829 2 View
hi, i have been advised to use methanol as my fixing agent for viewing tubulin staining. All this while i have been using PFA. I dont have problems getting clear staining. The question is, why do...
02 February 2017 6,095 0 View
recipe for 10% PFA: add 25g PFA to 250ml water . Heat at 50 degrees till dissolve. diluting 10% PFA to 4% PFA: 10ml of 10% PFA + 2.5ml 10X PBS + 12.5ul of 20% triton + 12.375ml H2O. sometimes i...
02 February 2017 8,772 2 View
hi im doing mechanical compression on cancer cells. i notice as expected they become flatten. the attached pictures from top refers to compressed, while bottom image refers to uncompressed. i was...
02 February 2017 7,484 0 View
i constantly see unfocused not sharp stainings. i did immunofluorescence on polyester transwell inserts. an example of the image below is one that shows alpha-integrin staining(green). the problem...
02 February 2017 7,259 0 View
If on day 1 i revived the cells and on day 2 its confluent enough to be seeded on 6 well plates. Can i do experiment on the 6 well plates on day 3? Or is it too early? I did spin down away the...
02 February 2017 6,597 2 View
if the culture was previously grown in media without penstrep, will a sudden switch to penstrep media kill the cells?
02 February 2017 7,229 1 View
So previously i grew cells as a confluent monolayer on transwell insert with pore sizes that prevents migration. Then once the cells are attached, i place a 5mm thick agarose-dmem agar cushion...
02 February 2017 7,192 3 View
first 2 images are compressed MDA-MB231, bottom most image is uncompressed control
02 February 2017 9,349 0 View
do you see any changes in actin staining patterns. I did mechanical compression on MDA-MB-231 cells. basically this involves growing cells as a monolayer on transwell insert, followed by placing...
02 February 2017 6,393 7 View
hi, i am interested in studying how mechanical compression alters the distribution of proteins known to be involved in sensing mechanical compression. For these proteins, how long do people...
02 February 2017 4,580 0 View
hi I observed such a-tubulin distribution changes in control and flattened(compressed) MDA-MB-231 cells. do let me know if you disagree or find other differences. 1) Upon compression, the...
02 February 2017 7,624 8 View
hi, i created this device which can mechanically compress cells. I did immunostaining of the cells and observe some apoptotic nuclear fragmentation of cells after compression. I was thinking of...
02 February 2017 7,273 6 View
i grew cancer cells on a transwell insert and then place an agarose cushion over the cells, followed by a weight on top of it. i notice that if the cells are more confluent (100 percent for...
02 February 2017 6,074 1 View
Do you see any abnormalities in alpha tubulin staining of MDA-MB-231 cells here? i actually compressed the cells. i hope to see compression mediated changes.
02 February 2017 6,080 0 View
Hi, I intend to grow monolayer of cells on transwell insert coated with collagen. I want to eventually extract RNA but will these cells invade and enter through the collagen(invasion) making it...
01 January 2017 938 1 View
im trying to compress breast cancer cells. Obviously, the variable that changes here is uncompressed and compressed group of cells. But the cells will be compressed against collagen coated gel on...
01 January 2017 6,534 1 View
hi i will be performing global transcriptome analysis of monolayer of cells exposed to mechanical compression. but before i do this, i will do staining on cells to look at effect of compression...
01 January 2017 5,116 1 View
Hi I usually perform immunofluorescence on cells grown on coverslips. But this time im growing them on transwell inserts. All the fixing and antibody staining/wash steps will be soaking the...
01 January 2017 8,336 5 View
Hi i did immunocytochemistry recently. For the fixation step i diluted the 10% paraformaldehyde to 4% by using water. I notice that my Mda-mb-231 cells become rounded when previously it is more...
01 January 2017 438 4 View
hi i am thinking of doing RNA-seq on cells that are mechanically compressed. but before i do that, i have been advised to maybe look at RT-PCR for some genes first. what are some good genes whose...
01 January 2017 6,221 3 View
hi so i was told that wherever possible, it is best to use warmed reagents when doing immunocytochemistry staining steps. In the below steps, which reagents must be warmed? is it safe to say that...
01 January 2017 9,232 4 View
i did mechanical compression on cancer cells and i notice this peculiar nuclei morphology in compressed cells. Any thoughts or opinion on this one? Is this blebbing? and is doing a nuclear lamin...
01 January 2017 1,085 3 View
i did mechanically compress cancer cells and looked at vimentin staining patterns. I am quite inexperienced, and could not detect any changes in distribution pattern. i will appreciate any...
01 January 2017 6,003 0 View
i did mechanical compression on cancer cells and then looked at actin staining pattern. I lack experience and did not see any changes in actin pattern. it could take a keen eye to detect changes...
01 January 2017 9,188 3 View
i did mechanical compression on cancer cells and then looked at a-tubulin staining pattern. I lack experience and did not see any changes in a-tubulin pattern. Do you see any changes? top image...
01 January 2017 1,694 4 View
hi do you see any changes in paxillin staining pattern upon mechanical compression of cancer cells? top image is compressed below image is uncompressed
01 January 2017 7,931 0 View
-opinions are needed
12 December 2016 949 0 View
hi i have been thinking of simulating the 'squeezing' metastatic cells have to go through as they slither through endothelial cells in intravasation. cells with high metastatic potential are able...
12 December 2016 5,989 2 View
i have a cell stretching apparatus. Was wondering if i could study mechanical response of metastatic cells to stretching but is there any step in the metastasis cascade in which stretching of cell...
12 December 2016 7,033 0 View
I have looked through several journals that employ this 6 well- transwell, growing cells on a permeable monolayer followed by placing an agarose layer and a piston on top to simulate mechanical...
12 December 2016 2,005 2 View
hi everyone, i hope to get some expert advise/opinion on this matter. Im a phd student tasked to come up with my own project and i have been reading a lot of papers. I am interested to know how...
12 December 2016 5,520 2 View
i have been reading some papers to get an idea of this. for example, i am aware that as tumor expand, the cells inside of tumor is exposed to compression forces. But there is a part in the paper...
12 December 2016 896 0 View
hi i have been honing my skills in western blot over the past month and have been getting faint bands despite loading 100ug total protein, and varying levels of antibody dilutions. my ponceu...
11 November 2016 4,431 8 View
hi im a noobie when it comes to lab work but recently i made a careless mistake and i dont think i can afford to throw it all away and make a new set of buffer due to lack of starting...
11 November 2016 7,049 4 View
Hi i would like to know the general way of indicating passage number to cells. I have heard that some people label the cell passage number as zero upon receipt from company. while some would just...
11 November 2016 2,767 5 View
I'm differentiating my cells which require temperature induction of 38 degrees for 14 days. I'm seeing weird stuff in my images. I hope the big cells are my differentiating podocytes which look...
11 November 2016 9,633 8 View
i am differentiating my podocytes cells which require temperature incubation at 37 degrees for 14 days. i notice that a certain portions of the plate the cells look different. the first attached...
11 November 2016 5,112 11 View
hi, i really need to look at a differentiation marker and for this i use a primary antibody derived from goat to detect synaptopodin protein. For my western blot analysis, i see a non specific...
11 November 2016 9,415 3 View
has anyone had to deal with cell lines that proliferate at 33 degrees and differentiate at 37-38 degrees (14 days needed) celcius? The cell line im dealing with are SVI podocytes (CLS). i...
11 November 2016 9,424 7 View
A: Protein lysis buffer components: what do you think of my lysis buffer preparation? 1) 1M Hepes (pH 7.5) stock: 11.915g hepes Add 45ml miliQ Adjust pH, top up with miliQ TO 50ml 2) 5M...
11 November 2016 5,522 0 View
Hi this may sound like a really stupid question but im totally new to lab work. If i purchase plasmids like the one below, SYNPO (GFP-tagged) - Human synaptopodin (SYNPO), transcript variant 3,...
11 November 2016 378 7 View
hi, is there a rough guide or from your experience, an estimation of cell density being translated to cell confluency? i need this rough translation for initial seeding density.
11 November 2016 1,296 4 View
Hi, I'm culturing immortalized mouse podocyte cell lines. Currently at differentiation stage. As seen in image below, the blue circled regions are cells that are undifferentiated, while the red...
11 November 2016 7,351 2 View
is it possible that any form of contamination in cell culture affect the morphology of eukaryotic cell in cell culture?
11 November 2016 4,879 0 View
hi i need a second opinion as to whether my podocyte cell lines are differentiating. these are SVI podocytes and they proliferate at 33 degrees celcius, differentiate at 38 degrees celcius. It...
11 November 2016 9,496 3 View
hi im a new researcher and is hoping for some opinion on this. i am working on podocytes cells, which are part of the glomerulus of kidney. They are therefore exposed to glomerular pressure, and...
11 November 2016 7,458 2 View
hi im a beginner in lab work. i have been doing multiple runs of western blot to detect protein (be it protein of interest or housekeeping proteins) but i couldnt get a good band. i realise...
10 October 2016 8,036 11 View
Hi im trying to detect by western blot a nuclear protein. I have been trying yo do this with no success until i realised that nuclear proteins may require different way to prepare and detect....
10 October 2016 9,994 6 View
hi i did western blot recently to detect synaptopodin (100kd) and actin (42kd) in mouse podocytes. The primary antibody against synaptopodin is derived from goat, while the primary antibody...
10 October 2016 7,545 18 View
i am aware that IF may not be that quantitative to compare expression levels but merely for detection (present or absent) is IF more sensitive than western blot? im habdling a cell line that...
10 October 2016 9,420 10 View
Hi one of my projects include looking at phenotypic changes upon knocking down of a certain protein. So i started first by looking for expression levels of this protein by western blot. However i...
10 October 2016 5,509 3 View
Hi, I´m a new researcher and I am trying to verify a particular cell line before I start experiments. I am looking at expression of certain key proteins specific to this cell line. I have been...
10 October 2016 6,009 6 View
hi im new to research so i will be happy to receive any advise on this matter. I have a new cell line with me and i am proceeding to ensure that this cell line is indeed what it is by detecting...
10 October 2016 5,170 3 View
hi im new to research and have been told to always prepare the 5% milk FRESH each time and not to reuse it for other days, ie: store leftovers at -20 deg and used on other days. but i realise...
10 October 2016 783 3 View
hi im a newbie when it comes to lab techniques. i have ttried detecting western blot expression of some proteins but i couldnt get any expression (even housekeeping genes). currently in the...
10 October 2016 7,683 11 View
i have 4 lanes worth of samples. it is always 1 lane away from the protein markers. it is at lane 2, 5, 8, 9 the amount of total protein lysate is 60ug. the first image is 10 seconds after water...
10 October 2016 4,388 3 View
hi i would like to know if sterility or accidental addition of microbes during immunofluorescence part must be observed. The steps starting from taking out the culture plates from incubator,...
10 October 2016 3,890 4 View
there are dots within the nucleus. no small dots outside the nucleus/cell. or are these just background?
10 October 2016 4,035 4 View
hi im working with podocytes. I am intending to perform IF on these cells. But one puzzling thing happened. 6hrs post splitting the cells into the plate meant for immunoflorescence, i notice the...
10 October 2016 3,618 6 View
hi there are numerous studies done conducting proteomic and phoshoproteomic analysis on certain cells exposed to mechanical stress. These analyses showed upregulation/downregulation of genes which...
10 October 2016 6,162 0 View
Plan for podocytes cell line: · 1 vial contains 1.5ml, to a total of 2.25 x 106 cells · Passage number upon arrival is 23. Each batch can be used until passage 40 Thawing from...
09 September 2016 6,941 1 View
here are the 4 protein markers: 1) WT-1 , rabbit source, 52Kd 2) synaptopodin, goat, 100kd 3) POPX2, goat, 54kd 4) actin, rabbit, 42kd I have heard about cutting the...
09 September 2016 5,004 5 View
hi i have learnt 2 slightly different protocols for lysing cells prior western blot. which method is better? method A: 1) Once cells reach 75 confluency in 60mm dish plate, remove media, and wash...
09 September 2016 3,227 8 View
hi i intend to study the phosphoproteome changes upon exposure of a glomerulus cell to increase glucose concentration. diabetic conditions have been shown to induce apoptosis in these cells in...
09 September 2016 8,543 2 View
hi I intend to expose my cells to mechanical stretching while in SILAC media. Im a pretty inexperienced researcher being an undergrad but what kind of downstream mass Spec analysis can i perform...
09 September 2016 1,387 2 View
hi I would like to know your expert opinion on this matter. I have been reading up on podocyte response to mechanical stretch. Most studies wanting to investigate genes upregulated/downregulated...
09 September 2016 3,916 1 View
- I plan to compare proteins coming from the control cells and cells exposed to mechanical stress. - I will grow these cells in SILAC media then harvest the proteins. - I intend to do a global...
09 September 2016 1,376 7 View
The cell line that i use proliferates at 33 degrees, and differentiates at 37 degrees. they are hardly any detailed protocols avaIlable online so i was wondering if this protocol i adapted below...
09 September 2016 2,447 14 View
hi, does anybody have a protocol on the maintenance, passage, seeding , differentiation of podocyte cell lines? the cell line im using is from endlich. if sharing the protocol here is hard,...
08 August 2016 1,461 0 View
im currently working on podocyte cell lines. protocol recommends use of accutase to detach adherent cells. can this be replaced by trypsin?
08 August 2016 6,975 3 View
Hi I am interested to find out if TGF-beta is involved in different cell-cell communication between 3 cell types in the glomerular filtration barrier. I have search quite a number of published...
08 August 2016 5,170 1 View
grasping at straws now. any experts in the field of nephrology willing to guide this PhD student who has read countless research papers but still could not find any research topics.
08 August 2016 3,399 0 View
Hi 1) may i know the best approach to elucidate the possible pathways coming from any membrane bound proteins. currently, the cytoplasmic binding partners of these proteins are not known. I'd...
08 August 2016 2,078 2 View
what kind of d-glucose source should i use? is there even a cell culture grade d-glucose or any grade will do?
08 August 2016 5,721 0 View
Is there a general rule to follow as far as number of nucleotides and the sequence is concerned? Or is there a strict rule to follow depending on restriction enzyme to be used? Buffer sequences...
07 July 2016 3,679 3 View
I accidentally added 2.5X the normal amount of oligoDT primers into my reverse transcription reaction. What are the chances i have screwed up my experiment?
03 March 2016 6,723 1 View
Hi I've been trying to introduce a nucleotide substitution into a minigene. My protocol includes PCR amplification (with minigene as template) using KAPA HiFI PCR kit with 4 different forward...
02 February 2016 5,375 7 View
Hi all, I am new to research and I am trying to learn as much as i can, i hope i can get guidance and advice here. I am doing a minigene transfection into HEK293T cells. I have done the RNA...
01 January 2016 8,592 10 View
