37 Questions 94 Answers 0 Followers
Questions related from Jaya Aseervatham
Hi, Im trying to use the image segmentation tool in image J for my image and Iam not getting it correctly to find out the co-localization of two proteins. If anyone has used it, could you please...
10 October 2019 6,965 4 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...
09 September 2018 6,614 4 View
Hi, I want to design a blocking peptide for the C terminus of my protein. If anyone knows how to do it, please help me out. Thanks jaya
05 May 2018 3,842 3 View
Hi, Iam encountering a problem in the cell culture. I have hela cells stably expressing connexin 36 attached to GFP on the C terminus. My problem is there is connexin 36 in the internal vesicles,...
12 December 2017 3,922 3 View
Hi, Does anyone have a protocol for native gel that is working for membrane proteins. Also I have a clarification to be made what is anode and cathode buffer. iam using the tetracell from biorad...
10 October 2017 4,852 2 View
Hi, Iam working with a protein which is attached to GFP and expressed in Hela cells. I want to do single particle imaging using cryoEM. Im trying to do an IP with GFP antibody and trying to use a...
10 October 2017 2,406 1 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...
09 September 2017 6,145 5 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...
08 August 2017 8,633 3 View
hi, I am working with GFP tagged connexin 36 transfected in hela cells. I do a crude membrane preparation and then used chaps to solubliize the membrane for two hrs and run it on a 10% gel. When I...
03 March 2017 9,129 10 View
Hi, Im confused on how to present my result for western blot for the paper. I did the experiments thrice by licor and quantified and for each time the machine gave me different values for the same...
10 October 2016 3,527 8 View
Hi, In continuation of my earlier post regarding the expression of my knockdown using lentivirus, I isolated the RNA, made Cdna and did the qpcr. All the reagents were from biorad. I have attached...
03 March 2016 9,244 3 View
Hi, Iam doing qpcr for a gene say X which has been silenced by shRNA. I used 1ug of RNA for the cDNA conversion and I took 1ul of the cDNA product for qpcr. When I look at the amplification...
03 March 2016 844 5 View
Hi, Iam starting a new project in which Iam silencing down two genes by lentivirus. Lets say gene x and Y. I have two single and one double knock downs. I want to study the expression of the genes...
01 January 2016 8,424 4 View
Hi, My lab has stable clones from OSC2 cell line having puromycin resistance by lentiviral transduction. I would like to know if antibiotic should be added to the culture medium after the cells...
10 October 2015 5,944 4 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...
09 September 2015 571 8 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...
07 July 2015 5,269 7 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....
07 July 2015 766 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...
07 July 2015 7,521 32 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...
03 March 2015 6,664 9 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...
12 December 2014 3,771 16 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...
12 December 2014 4,659 7 View
Hi, I am working with a mesenchymal cell line. My protein of interest is around 57 kDa and there is a published report on this cell line for the same protein. But the problem is, when I do the...
11 November 2014 9,658 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...
11 November 2014 5,068 17 View
I am using a stable clone which expresses my protein of interest, with G148 resistance and I use 500 ug/ml of G418 in the medium which is DMEM. Now I want to transfect with another plasmid having...
11 November 2014 461 5 View
Hi, Iam working on mesenchymal cell line and I have done a Co-IP for a target protein. Do I have to run the protein on native gel or can I do it using SDS-PAGE. Is there any difference between the two
10 October 2014 1,703 1 View
Hi, Iam using MC3T3 E1 cells for my experiment. For an experiment I was growing the cells for 10 days. Daily I used to collect the cells for western. On the 5th day, after I added the lysis...
10 October 2014 8,162 3 View
Hi, I am using a nuclear extract kit from active mottif to isolate the nucleus and using this for Co-IP using the thermofisher co-Ip kit, which uses the antibody immoblization. So my antibody does...
10 October 2014 3,232 11 View
I am using thermo scientific stripping buffer to strip the membrane for 45 -60 min.The first antibody is anti rabbit. But when I reprobe with the second antibody which is also antirabbit, the...
09 September 2014 627 6 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...
09 September 2014 2,384 4 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
09 September 2014 648 4 View
Hi, I am using C3H10 and MC3T3 cells both from mouse origin. Im using trans gene 9 transfection reagent for transfection. But the efficiency is very low. I am using 600ng DNA / well for 24 well...
09 September 2014 7,243 8 View
Hi, I would like to know if the RIPA buffer is different for SDS PAGE and for IP or Co-IP. If so what is the composition to be used. And do I have to run the samples after Co-IP on SDS PAGE or...
09 September 2014 2,776 7 View
I am working on mesenchymal cell line and I am trying to find out the time and dose response of colchicine to the cell line but not able to get the concentrations for cell sync. Any response will...
08 August 2014 4,992 4 View
Hi, I am expressing connexin 36 in Hela cells and want to perform a western and Co-Ip. Iam going to use maltopyranoside as the detergent. Has anyone out there used this and can you share your...
01 January 1970 6,293 3 View
Hi, Iam working on connexin 36 which is a membrane protien, and is GFP tagged. I did a western using 20mM maltopyranoside as the detergent, but wasnt able to see any band with the molecular weight...
01 January 1970 4,633 4 View
hi, Iam doing a western for a membrane protein with a mol weight of 36 kda attached with GFP. First I isolate the crude fraction were the membrane is present and then lyse with 20mM beta D...
01 January 1970 6,982 3 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 3,827 2 View
