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Questions related from Asa Wu
Dear All, I have question on choosing WB bands image for publication. My target protein has two forms: 1.full-length form (very strong signal) 2. cleaved form (faint/low signal) in the same blot....
05 May 2016 7,305 5 View
I have done IF with Abcam antibody, but the image that I get is not look like as the Abcam review on the same anitbody. My image (attached red image) is kind blush-like or blur color?, but from...
05 May 2016 8,198 3 View
Dear All, I tried to study co-localization of two different proteins by using immunofluorescence staining, since the western blot result of both protein shows significantly increase in nuclear...
04 April 2016 1,684 10 View
Dear All, I have SH-SY5Y grown on coverslip (precoated with 0.01mg/ml for 1hr at 37C) in 24 well plate. When the cells were ready, I wash it breifly with PBS, then fix it with 4% Paraformaldehyde...
03 March 2016 7,809 18 View
Dear All, I am study FasL protein expression using western blot in oxidative stress-induced cell death in SH-SY5Y. My results mostly shows that FasL expression is non-significant changed or...
03 March 2016 9,041 4 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...
02 February 2016 1,406 8 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...
02 February 2016 3,904 4 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...
02 February 2016 7,539 2 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...
01 January 2016 3,220 7 View
Dear All, I have a problem to do stat analysis to determine significant difference between control group and each condition group (vary dose of H2O2 treatment to cell line) of a target protein in...
12 December 2015 4,669 13 View
Hello, Both are free. From what I have tried, 1. Image Studio Lite from Licor is much easier to use, but it doesn't have lane background subtraction, which I think is the best method for...
12 December 2015 5,143 2 View
Dear All, When I developed the protein signal with ECL on PVDF membrane, it displays many scratch marks background on the membrane. The blot was covered in plastic sheet to prevent drying. These...
11 November 2015 6,603 0 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...
11 November 2015 820 5 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...
11 November 2015 2,892 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 3,993 1 View
Dear All, 1. In selecting area around a western blot band to analysis, Does it necessary to select all the band in same size of area? 2. In software such as Image studio, there are methods of...
10 October 2015 9,984 2 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...
10 October 2015 2,360 5 View
Dear All, From google search, there are three methods to do background subtraction in analyzing western blots with ImageJ? 1. Draw one horizontal line to cut all signal peaks from background or...
10 October 2015 3,677 18 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...
10 October 2015 4,707 9 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...
10 October 2015 3,958 3 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,...
10 October 2015 5,410 4 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),...
10 October 2015 7,125 14 View
Hello, On sigma website, there is Trizma/Tris buffer table recipe for mixing tris base and tris-hcl for specific pH range between 7.2-9.0, without need for adjust pH. I am looking for the recipe...
10 October 2015 9,007 3 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...
10 October 2015 9,147 2 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,
10 October 2015 4,363 5 View
Dear All, In experiment to treat cell culture with different substances in different point of time, for example the cell was treated with 10ul of solution A first for final concentration (f.conc)...
09 September 2015 2,801 5 View
Dear All, If I would like to subculture cell culture again after had been subculturing them on yesterday. Could it have any effect to cell healthy? Thank you Cheerio,
09 September 2015 7,420 3 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...
09 September 2015 7,964 4 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...
08 August 2015 4,718 8 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,...
08 August 2015 5,896 3 View
Dear All, Is it ok to leave membrane incubating with primary antibody (dilution with blocking buffer) at cold room (4°C) for a few day? I usually incubate primary overnight, but sometime it's on...
08 August 2015 3,537 15 View
Dear All, Many suggests that it is better to run SDS-PAGE in lower voltage, for example 100V in mini-gel, espcially to separate close molecular weight bands, but I don't understand what is the...
08 August 2015 5,247 13 View
Dear All, For new/nerver-used antibody and no recommend dillution buffer to use with in datasheet, Should I start trying to dillute it with TBST alone first? I know that dillue antibody in...
08 August 2015 2,652 3 View
Dear All, I google many guides and they suggest that larger proteins are more difficult and take more time to transfer from gel to membrane in western blot. Is it possible that some proteins,...
08 August 2015 8,435 5 View
Dear All, I have read many questions and answers in this website, and found out that some people mentioned a method to reprobe another primary antibody of different target protein without...
07 July 2015 5,218 7 View
Dear All, I would like to detect NFAT (130-160kda) and its loading control histone h3 (15kda) together on same membrane. However I have never transfered more than 100 kda protein before. Many WB...
07 July 2015 4,679 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...
07 July 2015 6,731 9 View
Hello Guys, I have some questions regarding to buffer and method for cultured cell fractionation as following: Can I just use PBS with some inhibitors for buffer? because what matter is centrifuge...
05 May 2015 607 2 View
Hello All, I am planing to do experiment on treating H2O2 (hydrogen peroxide) on SY5Y cell for 24 hr to determine concentration of different proteins, but before that I have to find the toxic dose...
05 May 2015 7,594 3 View
Dear All, I want lyse whole (including nucleus) SY5Y cell without mechanical disrupting for Co-IP. I have read many resources, and it can be done by either adding 0.1-0.25% Sodium Deoxycholate or...
05 May 2015 255 3 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...
05 May 2015 632 3 View
TritonX-100 Lysis buffer recipe = 25 mM Tris•HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 5% glycerol I am sure it won't break up nuclear membrane but not sure whether it can break up...
05 May 2015 9,468 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...
05 May 2015 1,506 4 View
Hi All, 1. What voltage and how long do you run to get the best band resolution? 2. Is it necessary to run low voltage first before changing higher voltage? How much voltage is considering low...
04 April 2015 1,684 0 View
Hello All, Both abcam and biorad protocols state that low methanol concentration in transfer buffer could swell gel pore and this can increase transfer efficiency of protein. So I just wonder that...
04 April 2015 8,233 5 View
Could you kindly help me to specify what could be possible cause of my problem with band in Western blot as showed in my attach picture? picture 1 is 40 kda, picture 2 is 60 kda. 1. Some time, the...
04 April 2015 626 16 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...
04 April 2015 2,194 5 View
Hello all, When doing cell subculture, many guides suggest to passage cell at ~80% of cell confluence. But if I want to collect treated cell sample, should I collect the cell at ~80% of cell...
03 March 2015 3,893 7 View
Hi All, I want harvest SH-SY5Y cell and collect the protein sample to do western blot. There are many available method for detaching the cell, such as trypsinization, scraping in RIPA buffer, and...
03 March 2015 2,614 17 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...
03 March 2015 6,741 4 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...
03 March 2015 8,833 12 View
This is very good method to prevent gel leakage when casting the gel, but I found out that when running the sample in gel. The sample can only go down max and stuck at around 1cm above the high...
03 March 2015 1,679 0 View
Dear All, I am doing experiment to see effects of treating cell culture with different chemical substances. I have to treat the cell with liquid substance A with specific concentration first...
03 March 2015 1,166 5 View
Hi All, I wonder that if I have dilute a very small volume of substance A in a very large volume of substance B. For example dilution 1:20000 of an antibody in TBST. Mixing it well by vortex or...
03 March 2015 1,593 2 View
Hi All, I do store the membrane in a box filled with PBST, but the proteins on the membrane seems disappeared through time (after a few week or a month). Is there a better way to store the...
02 February 2015 629 12 View
Hi I just wonder whether boiling protein lysate in RIPA buffer before storing at -80°C can keep the protein last longer. How long can it store? Thank you
02 February 2015 3,099 2 View
Thank you.
12 December 2014 3,000 22 View
Hi All, When gel casting is finished (polymerized), the line between stacking gel and separating gel shows double line reflection. It seems to be reflection than another layer. What is the cause...
12 December 2014 8,316 1 View
Hi all, I have tried many method such as parafilm, 5% agarose. My gel is still leaking. I have read the top comment on the youtube video (https://www.youtube.com/watch?v=b45nSOyPP_4). He suggested...
12 December 2014 343 17 View
Hi, I just wonder if I make stock solution of resolving and stacking gels by adding such as 30%acrylamidemix, 10%sds, and Tris8.8 or 6.8. Can I store it at 4 °C, and for how long? Have you every...
12 December 2014 7,444 3 View
Hi All, Is it ok to prepare stacking and seperating/resolving gel stock solutions (mixing between Tris, 30%acrylamide mix, 10%SDS, and water, without adding temed and 10%aps) and store at 4...
12 December 2014 4,275 3 View
Hi all, I read many protocols and Q&A in ResearchGate. Many can run protein SDS-page electrophoresis at 200V constant using same set of equipments that I use (Bio-Rad Mini-PROTEAN® Tetra Cell...
11 November 2014 9,614 3 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...
11 November 2014 3,862 4 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...
11 November 2014 3,859 3 View
