Does anyone know how to use image segmentation in image J?

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...

09 October 2019 6,877 4 View

Anone facing phototoxicity in IF samples?

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...

08 September 2018 6,532 4 View

Anyone working with connexins?

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,...

11 December 2017 3,842 3 View

Help with cleaving GFP from the protein of interest ?

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...

09 October 2017 2,326 1 View

Protocol for native gel for membrane proteins?

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...

09 October 2017 4,759 2 View

How to represent western blot for paper ?

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...

08 September 2017 6,061 5 View

Is anyone working with halotag proteins?

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...

07 August 2017 8,554 3 View

Help with western blot. hello any one there?

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...

02 March 2017 9,048 10 View

Should I give the SD in the fold difference and the P value for western blot for publicaiton?

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...

09 October 2016 3,329 8 View

How much of cDNA to use for qPCR?

02 March 2016 375 5 View

Why does blocking with my immunogenic peptide cause nuclear fluorescence in ICC?

Hi all, I am validating a polyclonal antibody raised against human ANT4, through WB and ICC. In most of my controls, the antibody shows very little off-target binding, but when I pre-incubate the...

03 March 2021 5,999 3 View

No title

23 February 2021 4,014 3 View

How to reduce static electricity of powder after lyophilization?

I am working on synthesizing and then purifying peptides using the SPPS system. After purification of the peptide, the peptide powder is obtained using a freeze dryer. When measuring the powder,...

22 February 2021 10,099 5 View

Can we remove the fmoc group after peptide cleavage?

I have synthesized the peptide using Fmoc based solid phase peptide synthesis. Also I have cleaved the peptide from the resin and lyophilized it, but later realized that, I forgot to deprotect the...

21 February 2021 4,537 3 View

How T cells recognise non-peptide antigens?

This might sound very basic question, but please share with me an explanation of how the T cell is activated when the antigen is not a peptide? What happens? And what are the molecules involved in...

21 February 2021 564 3 View

Help with Amyloid beta detection protocol (western-blotting)?

Hi everyone, I am a first year PhD student working on amyloidosis. I would like some tips and tricks to crack efficiently my current nightmare, i.e. the western blotting of the Amyloid beta and...

11 February 2021 2,513 3 View

How to assing secondary structure to calculated peptide/protein having xyz file?

Hi, I have an optimized structure of some peptides and I would like to represent it as a secondary structure - cartoon. Ok, so server apps ask for pdb file (like Stride). I tried in different...

04 February 2021 517 3 View

Identical spectra for L- and D-peptides after they self-assemble?

Hi dear all, I need advice. I have two peptides with the same sequence but one consists of L-amino acids only and the other consists of D-amino acids only. when they self-assemble in aqueous...

03 February 2021 2,032 3 View

FLAG purification, elution doesn't have protein?

I am purifying a heterotetramer, with one subunit having a 1D4 tag and the other having a FLAG tag. I purified from Sf9 with HBS pH7.3 and 0.002%LMNG first with Anti-1D4 resin and got an adequate...

02 February 2021 3,860 1 View

How long can synthesized peptides using SPPS be stored? Storage Conditions?

I have been synthesizing peptides on resin beads. I am wondering what best storage method is there to store the beads after synthesis before cleaving. And how long can they be stored in that...

02 February 2021 5,636 5 View