Cytoskeletal Proteins - Science topic

It has been shown that endonuclease can cleave DNA to cause apoptosis, while activated calpain can hydrolyse cytoskeletal proteins, including spectrin, fodrin, actin and tubulin. If the actin will be hydrolysed, Why we always choose beita-actin as the marker when we use western blot to semi-quantify some protein. Does it make sense?

For my in vitro binding experiments with G -actin , I need to prevent spontaneous F-actin formation and maintain G-form. Please suggest how I can do it.

Hello!

I am investigating how cytoskeletal proteins change localization under detached conditions. I've seen that cells can remain viable on soft agarose (1% noble agarose in PBS and DMEM) for 24 hours, but removing them without getting a lot of agarose as well is tricky. Does anybody have any tips or suggestions for this?

Thanks!

I wish to measure plasma concentration of erythrocyte membrane lipids and proteins in Theileria equi-infected erythrocytes, getting a detailed protocol from literature search had been challenging

Hi,

I actually work on heterologous protein expression in yeast. my purpose is to secrete protein normally present in the cytosolic compartment. Due to absence of any signal protein on the SDS-PAGE gel , I'm seeking for specific sequences that could explain why my proteins of interest are not present in the culture medium. After examining precisely the nature of the sequence, I've identified sequence motif that could belong to Endoplasmic retention signal, which could be very surprising for cytosolic protein.

Does anyone have experienced that issue.

Thanks for our answer

Ezrin is a FERM domain cytoskeletal protein that in the inactive state assumes a conformation by which the N-terminal domain binds the c-terminal actin binding tail. I know phosphorylation leads to protein to open up and bind to actin filaments. How can I design a biosensor to detect activation state of Ezrin? what are the positive and negative control constructs?

I am looking for a paper showing evidence of the effects of DMSO over the polymerization of actin.

i want to detect my his tagged protein in the yeast culture supernatant using western blot, i want to use loading control but i'm little confused about which loading control i should use. i already tried with GAPDH and Beta-Actin but i didn't get the band but i got the band for HIs antibody (my target proteins).  

I will detect the function of flourescent tagged G13 protein by using  RhoA G-LISA Activation Assay in hek 293 cells.First flourescent tagged G13 protein will be transfected to hek cells then  I'll applied  LPA to stimulate G13 and Rho-GTP level will be detected with this assay kit. The hardest part is when I prepare cell lysate, I dont know how much ng g13 plasmid, how much uM LPA should be applied to how many hek cells.These parameters are changable due to cell types and I couldnt find this type of research with hek cells. İf I try different amounts of combinations, I will need at least 2 or 3 assay kit which means a lot of money.Do you suggest any referance research?

Using either MDCK, ARPE-19, or pig RPE cells. 

I have no other astrocyte markers that GFAP, but I am being required for an additional one because "GFAP is a promiscuous antibody". However, I have SR101 and I'd like to know if that would be an acceptable control to identify astrocytes in cell culture.

I use commercially available Qdot secondary antibodies that work in general. However, compared with e.g. Alexa488 labeling in the same sample I do not get the same specific staining, i.e. actin stress fibers are barely visible. The staining looks a bit spotty and there is not a very high degree of colocalization between Qdot/Alexa.

I tested a range of fixation/permeabilization methods (Acetone, methanol, formaldehyde, TritonX100 hi/lo), because I assume it is an accessibility problem. This is what the LifeTech Support suggested and what I somehow saw in a more or less good microtubule staining - there, at the thickest part of the cell, the microtubule Qdot staining was weaker compared to Alexa. I also tested embedding media (Cytoseal 60 vs. Mowiol) and dehydration before embedding. Everything without success so far...

Any other ideas or working protocols?

Thanks in advance!

Hi Everyone

I'm currently working on proliferation and doing cellular fractionation and I'm finding hard to choose a cyto marker for my blots. I'm currently using tubulin or actin but these appear as a double band instead of the usual one, there are a few papers that show this. I've search r and sometimes they use kinases, but in my case these alter significantly, so I can't use them.

Any thoughts on alternatives or is tubulin or actin ok?

Thanks in advance

MMP-2 that I used are from peprotech and novoprotein. The sequence that has to be cleaved is a MMP-2 substrate that links two fluorescent molecules.

I use 20 nM HEPES, 150mM NaCl buffer with 100uM ZnCl2, 2mM CaCl2 but no cleavage occurs. MMP-2 is at 30nM concentration and the protein of interest at 5uM.

Years ago we developed a system of a new class of antibiotics that have a protein of a bacterial cytoskeleton as the target (s. our patent WO 2002087554 A 2). Are there other reserach groups that also work on the development of antibacterial agents with the target being a component of a bacterial cytoskeleton?

Cultured cells, endothelial, DNA has to stay intact for qPCR.

Looking for selective protein markers for cytosolic and mitochondrial fraction. It would be big help if someone can suggest a working antibody too for those proteins.

I would like to perform cytoskeletal staining using F-actin phalloidin. I just wonder if the 4% paraformaldehyde (paraformaldehyde in PBS) has to be prepared fresh? Is this really necessary? I really hope experienced researcher can assist me on this.

Want to visualize microtubule dynamics in live cells. Transfecting HeLa cells with GFP-Tubulin (Addgene) using Jet PEI (4h transfection — image 24h later).

Problem: GFP looks cytosolic without a sniff of a tubule.

Background: used 1-2ug plasmid per well of a 6-well plate. Cells are not screaming bright (on the contrary).

Am I missing a point here? The cells are *not* synchronized (cell cycle) but I would expect a population of different stages.

I thought this would be easy, so I am probably being stupid. Any help gratefully received. Thanks.