46 Questions 36 Answers 0 Followers
Questions related from Asa Wu
Dear All, I try to using ImageJ to merge cell images of red, blue, phase contrast which are RGB image type. According to some instruction, I have to convert them into 8/16 bit before merging them,...
20 June 2016 4,222 3 View
Dear All, I did IF staining adherant cell line on coverlip to see nuclear translocation of target protain (red fluorescent dye, Alexaa fluror 546) coutering with DAPI. I would like to know how to...
24 May 2016 5,998 7 View
Dear All, I recently started doing immunocytochemistry/immunofluorescence and I found that removing/picking up coverslip from 24-well cultured plate is very difficult, before I can place it to a...
14 April 2016 2,564 12 View
Dear All, I am looking for plastic/glass chamber for staining immunofluorescence in adherant cells and use confocal microscope to study localization of protein between nuclues and cytoplasm in...
14 March 2016 5,026 7 View
Dear All, I just switch from SPSS to Graphpad prism 6. In analisys, there is option to select experimental design between RM (repeated measure) and Ordinary (no pairing) for one-way ANOVA. What is...
25 February 2016 3,647 3 View
Dear All, Normally Fas ligand express in T-cell. I would like know how Fas ligand WB signal generally express in general cell line? Does it strong like actin or GADPH? i am plaaning to to measure...
24 February 2016 4,841 2 View
Dear All, I am real newbie in protein field. I would like to ask you a few question regarding to select band to measure in western blot. In case that blocking is fine and I use single primary...
15 February 2016 6,403 7 View
Dear All, In my western blot experiment, I have problem with blotchy blackground (like in attachecd picture) on the blot, which usaully appear when I expose low signal band for 15-30 min or more...
10 January 2016 7,228 6 View
Dear All, I use Graphpad Prism 6 to analyze my 6 sample group's data (N=3) by using one-way ANOVA with Turkey comparison rest. The multiple comparison result doesn't show any significant between...
23 December 2015 7,429 4 View
Dear All, To prepare protein sample to determine its concentration by Bradford protein assay, I usually take a few extract samples from sample group to dilute in different dillutions, then adds...
07 December 2015 2,642 6 View
Dear All, I try to make 5x Laemmli buffer (10% SDS, 50% glycerol, 25% 2-mercaptoethanol, 0.02% bromphenol blue and 0.3125 M Tris HCl, pH approx. 6.8.) adapting from Sigma's 2X Laemmli buffer, but...
21 November 2015 6,298 4 View
Dear All, My target protein has two isoforms, one is at 60kda, which give strong WB band, however another is at 45-48kda, which has very weak WB signal, even after 1hr exposure with GE's Amersham...
20 November 2015 6,092 1 View
Dear All, I have SH-SY5Y cells which has been 100% confluence or over (stationary or decline phases) in the culture vessel. The cells start to go oval and detach, but some of them are still...
11 November 2015 5,421 1 View
Hi guys, From my expirence there are pros and cons of with or without plastic sheet cover: 1. Plastic sheet cover sheet is good to keep the blot wet, good for long time exposure, but it will have...
27 October 2015 6,085 4 View
Dear All, According to atcc.org, SH-SY5Y cells have a reported saturation density greater than 1x10^6 cells/cm2. If that is true, one well of 6-well plate has surface area around 9.5 cm2,...
21 October 2015 9,843 4 View
Hello Guys, Vortexing cell suspension for short time (~10sec) to homoginize solution before distribute cell into vessels. Can this damage the cell? Thank you Cheerio,
20 October 2015 8,541 6 View
Dear All, In my experiment, I need cell to be 50% confluent in 100mm dish to treat drug and collect samples after 24h. To prepare cell test group (I use large number of cells. 8 of 100mmdish),...
15 October 2015 4,678 13 View
Hi All, When I subculture the cell from one vessels (flask) to new multiple vessels (six-well, petri dishs). I often have one or two culure vessel that has less or more cells than the others. I...
15 October 2015 5,380 3 View
Hello, Is it ok to prepare lysis buffer with adding protease or phosphatase inhibitors and store at -20ºC or -80ºC for later use, may be weeks or months, instead of adding protease or phosphatase...
14 October 2015 3,764 5 View
Hello, I just wonder that if SH-SY5Y cell will lose viability or starting to die after it reaches 100% confluent in the culture and culture media is still keeping renewal/replaced. Normally the...
09 October 2015 3,840 2 View
Hello, I would like to treat cell culture with some drug for 12-24hr, then collect its protein sample to study cell signaling response. Should I collect the cell culture when it is still within...
08 October 2015 4,262 8 View
Hello, For using ImageJ to analyse gel image, normally I select each lane for each rectangle/box. but some guides suggest to draw a rectangle around all the lanes of gel image in one box and then...
07 October 2015 8,195 5 View
Dear All, What is the ratio of FBS to trypsin to inactivate/neurtrilize trypsinization in cell culture? In some protocol, the ratio of 10%FBS media to 0.25%trypsin-EDTA is 1:1 or 2:1. ATCC guide...
03 September 2015 3,943 4 View
Dear All, I am plainning to use hydrogen peroxide to treat to cell and see its protein signaling response. Most of reference articles use Sigma's hydrogen peroxide solution. Unfortunately,...
28 August 2015 9,092 3 View
Dear All, My recipe of 5X SDS-PAGE sample buffer without beta mercaptoethanol is 156.25 mM Trish-HCl, pH 6.8, 62.5% Glycerol, 5% SDS, 0.025% Bromophenol Blue. How long should the sample buffer be...
14 August 2015 3,663 3 View
Dear All, I just wonder that is possible that some protein might not transfer to some type of membrane in Western Blot. for example protein A might transfer to only nitrocellulose, not PVDF. I...
12 August 2015 9,245 7 View
Dear All, My Lysis buffer recipe is 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100 and 5% glycerol. I would like to use this buffer to lyse whole cell, except nuclear membrane,...
04 August 2015 2,257 4 View
Dear All, I am planning to study the effect of melatonin or H2O2 on different treating time periods to the cells, for example, treating at t0 (starting time) and t12 (after 12hr), then collect all...
29 July 2015 6,144 8 View
Hello All, I am planning to collect nuclear protein and cytosolic protein from the same cell sample, and I need to find protein extraction protocol. The cytosolic protein is needed to do Co-IP. I...
21 July 2015 4,588 3 View
Dear All, Which company/brand provide the best bead or IP/Co-IP kit? In addition, I would like to ask you help about how to eliminate heavy/light chain signal. I am using Dynabead right now. It is...
19 May 2015 5,099 7 View
I would like to find cell lysis method to solubilize/break-up nuclear membrane without breaking/denaturing protein-protein interaction of protein complex in nucleus before doing CO-IP. I need to...
14 May 2015 6,288 3 View
If the protein A (60kda) and protein B (30kda) can form a protein complex together, is it possible to detect protein A band at 30kda (protein B location) when do western blot (incubating only...
09 May 2015 9,652 3 View
Hello All, I have the pellet that left over from lysing cell with Triton X-100 lysis buffer. Because I didn't sonicate the lysate and Triton X-100 lysis buffer cannot solubilize nuclear and...
04 May 2015 7,503 2 View
Hello, Some said that the sonication can break nuclear membrane and get the nuclear protein. However many protocol also use the sonication method to disrupt cell before isolating mitrochondrial...
03 May 2015 8,450 3 View
Hello Does NP-40 or Triton X-100 lysis buffer lyse/break mitochondrial and nuclear membranes to release out their proteins? In addition, RIPA can rip off all organelle membranes and RIPA = NP-40...
03 May 2015 635 6 View
Hello, I incubate two type of antibodies A and B together in the same membrane. Both are same species monoclonal, but bind to different protein molecular weight. It usually give me the result in...
02 May 2015 7,094 4 View
The dynabead protocol suggests to use at least 100ul of antigen-containing lysate to resuspend the bead in immunoprecipitation. I usually 10ug of lysate dilluted in PBS to get 100ul and give me...
24 April 2015 5,341 5 View
I would like to try to degas polyacrylamide gel 15 min before casting. But I don't have vacuum chamber or strong aspirator to put the gel in. Is there other way to degas the gel solution? Thank you
12 April 2015 7,658 2 View
Some protocol adds reducing agent to Laemmli sample buffer and store it at -70 or -20 degree Celsius. Some adds it fresh before boiling with protein sample. Have you guys ever tried both add...
07 April 2015 4,146 5 View
Hello All, Is it possible to re-developing signal without stripping in Western blot? and How do you do it? Please share. It has been stuck in my head for a while. My objective is just to...
20 March 2015 5,423 12 View
Hello All, I would like to remove only just ECL and leave most of primary and secondary antibody on the membrane, then store it in -20C for later re-developing the signal with new ECL, may be in...
13 March 2015 5,885 3 View
Hello Guys, My lab usually leaves cell to attach on cell plate overnight after cell subculture, but we don't know how long it usually take to attach on the plate. Do you know how long? Could it...
12 March 2015 6,699 6 View
Hi All, I have read a protocol that say it is possible to cast separating and stacking gels without intermediate steps requiring overlay water. They mentioned adjusting the glycerol percentage in...
03 December 2014 8,564 4 View
Thank you.
01 December 2014 5,306 21 View
Hi all, I tried using 15% gel SDS for running vertical SDS-PAGE gel electrophoresis at conditions: 100V, then 120V after loading sample is lining. I found out that the loading sample/dye (mixing...
27 November 2014 4,256 3 View
Hi All, I have been wondering for while about how long can store loading sample after mixing with loading dye, then boiled for 5 minute and at what temperature 4°C or -20°C for using in gel...
27 November 2014 6,786 3 View
