29 Questions 74 Answers 0 Followers
Questions related from Jaya Aseervatham
Hi All, I would like to know if there is a way to superimpose serial sections of tissues after doing immunofluorescence. The problem is I want to see the co-localization between few proteins and...
23 September 2020 1,305 3 View
hi, Is there anyone out there who has used R18 inhibitor peptide for inhibiting 14-3-3 in vitro. If yes could you let me know the concentration and does it need any peptide transfection reagent...
05 November 2019 3,687 0 View
Hi, Iam doing IF in HeLa cells and facing a weird problem. I use the standard protocol and when I expose the cell to laser light in the 488 channel, all the fluorescence fades the instant its...
30 September 2018 2,700 3 View
Hi, I am trying to represent my western blots for paper and have a clarification how to represent the Y axis in the graph. I normalized the test protein with actin and then divided my test protein...
28 September 2017 8,022 4 View
hi, Iam working with a halotag attached to my protein expressed in Hela cells. I was trying to pull down the halotag with magne beads and got nothing in the western. I tried increasing the...
23 August 2017 4,737 3 View
HI, Iam using licor rabbit secondary antibody green color in the 800 channel and I see a lot of background for some of the primary antibodies, though some of them work perfectly. Is there any...
23 June 2017 6,139 3 View
Hi, Iam using GFP nano trap from chromotek for my experiments and I want to study the structure of my protein by electron microscopy for which I need to get the protein in the pure form. After...
07 June 2017 1,677 2 View
Hi, Iam using the GFP Trap MA from chromotek for Ip of my protein. I followed everything as per the protocol, but was not successful. My protein was seen in the flowthrough, which means that it...
12 April 2017 4,750 21 View
Hi, I want to do a Co-ip of a connexin and its associated proteins for mass spec analysis. The problem is, to get the connexin and the bound proteins intact. Can anyone suggest a lysis buffer/...
07 January 2017 6,207 3 View
HI, I am doing a lentiviral knockdown of a gene called DSPP and have prepared the stable clones which took me about 4 month with puromycin selection. The problem that I am facing is, when I do a...
24 June 2016 9,428 4 View
Hi, can anyone tell me the calculation for MTT assay. Iam going to use triplicates of my sample and control in a microplate reader. I have untreated and treated samples. Do I have to have a...
02 April 2016 2,037 3 View
Hi, I have to do transduction using lentivirus and I have a doubt how to calculate the MOI. I have 5x10 3 ( 5000 viral particles/ ul). How much do I have to take to get MOI of 1 if I plate 10000...
28 January 2016 2,409 13 View
HI, I am working on breast cancer and I\m trying to use MCF12F as normal breast cell line. I purchased it from ATCC and Iam having problem growing it. It dosent seem to attach and the few cells...
29 September 2015 3,269 7 View
Hi, I would like to know if any one has tried formaldehyde for fixing vs PFA. I would like to know the difference in the primary and secondary binding when both the reagents are used. Jaya
07 August 2015 991 11 View
Hi, As the follow up of my earlier question about the negative control showing fluorescence, Ihave found out that the secondary conjugated Alexa 488 is showing the fluorescence in the cells. I did...
29 July 2015 4,606 32 View
Hi, Iam working with a cervical cancer cell line and Iam supposed to do immunofluorescence for MMP's. Iam facing a wierd problem. When I do the negative control with IgG, (Alexa 488) there is a...
23 July 2015 8,211 6 View
Hi, Iam working on a oral cancer cell line and Iam facing a problem. The DNA damage shows apoptosis but the caspase 3 and 9 levels are low. Is this possible or what Iam seeing is an artifact....
11 July 2015 4,508 10 View
Hi, Iam trying to study CDT1 and Gemenin, for G1 and G2 phase of cell cycle. Iam not able to get clear signals in the nucleus by immunofluorescence. Iam fixing my cells with 4% PFA. This this good...
17 March 2015 8,214 3 View
Hi, I am having a stable clone having G418 resistance and puromycin resistance. After expanding the cells, I freezed using 50% FBS, 40% medium and 10% DMSO, left the cells in -80 and then froze in...
08 March 2015 7,729 8 View
Hi, I would like to know, if there is a way to find the interactions between two proteins. Iam working on a dental protein called Dentin Matrix protein and loss of this activity is reported to...
13 January 2015 9,505 3 View
Hi, Iam working with the expression of Fgf 23 in osteoblast and cDMP1. invivo published results prove that cDMP1 inhibits Fgf23. But when I studied the promoter activity of fgf23 invitro using...
10 January 2015 3,891 16 View
hello, I am facing a problem with stable clone. I think I am having a problem but not so sure. I have c3h10 cells which is a stable clone with G418 resistance and the stably expressing protein is...
01 January 2015 8,188 4 View
hi, Iam facing a weird problem after transfection. I am using C3H10 meschencymal cell line which is a stable clone expressing a protein which is GFP tagged. Iam using this cell line to transfect...
15 December 2014 4,019 15 View
Hi, I am having bacterial contamination in cell culture. I see these wriggling at high magnification and as black balls at low magnification. My problem is, I see these only after I start...
11 December 2014 5,375 6 View
Hi, I am using an antibody which is indigenous. The problem is it binds non specifically to more proteins. I see around 8-9 bands on the western. Even if I use more dilution this same pattern is...
02 November 2014 7,631 16 View
Hi, I am using MC3T3E1 cells ( mouse preosteoblasts) for transfection with Xtreme9 reagent. 24 hrs after transfection I see lot of cell death. Is there a way to minimise it? Thanks
19 September 2014 2,000 3 View
Hi, I am using nocodozole (20-100ng) and colchicine (0.5-2.5uM) on C3H10 cell to arrest the cells in G2/M phase for 18-24 hrs. Then I do a western for cyclin B1. What I found was that there was no...
19 September 2014 3,270 3 View
Hello, I am trying to isolate the nuclear protein from mesenchymal cell using the nuclear isolation kit from Active mottif and then using this for Co-IP. I have tried many times, but Iam not able...
19 August 2014 4,030 4 View
Hi, Iam using donkey antirabbit secondary antibody at the 700 channel to detect my protein of interest (1;1000 dilution primary ). My problem is that the green channel is showing non specific...
01 January 1970 3,318 2 View
