Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!

Gangwolf's Content

There have been 41 items by Gangwolf (Search limited from 21-April 20)



Sort by                Order  

#132727 Secondary Antibody Problem

Posted by Gangwolf on 12 April 2012 - 08:00 AM in SDS-PAGE and Western Blotting

If you blocked with milk, anti-goat antibody might react with that.



#131861 which protein can I use as a loading control for nuclear protein fraction?

Posted by Gangwolf on 28 March 2012 - 06:12 AM in SDS-PAGE and Western Blotting

We frequently use TATA binding protein for nuclear extracts as loading control. You can also check if you have cytosolic contamination in your nuclear fraction by probing for A/B tubulin, as tubulin should not be present in the nucleus.



#131860 Best phospho-ERK antibody

Posted by Gangwolf on 28 March 2012 - 06:05 AM in SDS-PAGE and Western Blotting

Try this one http://www.scbt.com/...4-antibody.html
Way cheaper than Cell Signaling and it works great!



#131489 Getting too Many Bands on my Western Blot

Posted by Gangwolf on 22 March 2012 - 02:34 AM in General Lab Techniques


Sometimes loading too much lysate also causes multiple bands.
I don't know what you did prior to lysing your cells but using trypsing to dislodge the cells could definitely give you multiple bands as trypsin might digest your protein of interest.

This should only happen if you are looking at cell surface markers (vimentin isn't one), don't neutralise your trypsin (by adding FBS containing medium), don't wash the cells before lysing, and don't include protease inhibitors in your lysis buffer.

I routinely use trypsin to lift my cells for western blots and don't have any problems with multiple bands from tryptic digestion.


Respectfully, it is not entirely true!
In the past when I worked with a transcription factor and did what you suggested that one should do when using trypsin, the transcription factor was still seen as multiple bands until I stopped using trypsin.
Nowadays, I just lyse the cells directly on the plates/flasks.



#131417 Getting too Many Bands on my Western Blot

Posted by Gangwolf on 21 March 2012 - 06:50 AM in General Lab Techniques

Sometimes loading too much lysate also causes multiple bands.
I don't know what you did prior to lysing your cells but using trypsing to dislodge the cells could definitely give you multiple bands as trypsin might digest your protein of interest.



#130999 experimental design

Posted by Gangwolf on 14 March 2012 - 07:50 AM in General Lab Techniques

Please share =)



#130831 Detach cells and stop stimulation simultaneously- How?

Posted by Gangwolf on 12 March 2012 - 01:42 AM in Cell Biology

Thanks bob1!
Just for the sake of completing this thread, I’ve decided to do a cell surface receptor biotinylation assay. Basically, incubating cells with biotin (in cold PBS) which binds to all available primary amines on the cell surface. After 1h of incubation, un-bound biotin will be inactivated by incubating the cells in 50mM Tris pH 8 for 5 min. After that, I will lyse the cells and precipitate surface proteins with streptavidin agarose. Finally, the cells surface receptor will be visualized through western blotting.



#130731 Compartmentalization of signal transduction cascades

Posted by Gangwolf on 09 March 2012 - 05:41 AM in Research Idea, Design and Collaboration

Here are some examples http://www.sciencedi...097276509006777
http://www.ncbi.nlm....les/PMC1800644/



#130689 Help with Dual luciferase assay

Posted by Gangwolf on 08 March 2012 - 05:53 AM in Cell Biology

I had the exact same problem. Done an assay a million times and then one day it just stopped working. I still don't have an explanation to the problem but it helped when I used new cells and changed transfection reagent and this also meant that I had to re-optimize the assay.
Just do a simple experiment only using the renilla construct. Transfection with increasing amounts of renilla and determine the level where it reaches the saturation point (e.g increasing renilla= increasing signal to a point where it only levels off). Do this with all the construct and finally do the experiment with the amount of DNA that does saturate the signal.



#130205 Detach cells and stop stimulation simultaneously- How?

Posted by Gangwolf on 01 March 2012 - 02:35 AM in Cell Biology

Thanks for the answer, bob1!
Since my interest is to quantify the receptor expression on cell surface, is it possible to do this through western blot.
For example, I could stimulate the cells for 5min and then stop the the reaction with cold PBS and putting the plate on ice. While incubating on ice, I would add primary antibodies against the N-terminus of the receptor and after X minutes, wash off the antibodies and lyse the cells.
Transfer the cell lysate to eppendorf tube, add protein beads to immuniprecipitate antibodies bound to my target and subsequently do a western blot of the IP product.

How does this sound?



#130116 Detach cells and stop stimulation simultaneously- How?

Posted by Gangwolf on 29 February 2012 - 12:12 AM in Cell Biology

Hi all!
My purpose is to do a FACS analysis of a receptor tyrosine kinase on the cell surface of an adherent cell line. I want to do the analysis when the receptor has been ligand stimulated for 5 min. What should I do to stop the reaction at 5 min?
Should I add cold PBS and put the cells on ice and then scrape them of the plate?



#130073 Carrier protein concentration

Posted by Gangwolf on 28 February 2012 - 05:54 AM in Cell Biology

This should not be a problem!



#126826 Transfection problem after transformation!

Posted by Gangwolf on 10 January 2012 - 02:42 AM in Cell Biology

That I haven't tried! The plasmid that works great before transformation is not linearized, so do you really think it would make that big of a difference for transient transfection?



#126823 Transfection problem after transformation!

Posted by Gangwolf on 10 January 2012 - 02:18 AM in Cell Biology

I have done that Posted Image
Transfection efficiency is around 70% with GFP.
The expression of my construct at mRNA level is high and the construct that cannot be expressed is at the same level as my negative control empty vector.
In terms of stability, my protein can be detected by WB a week after transient transfection.

I have compared Lipofectamine, Turbofect, Exgen and JetPEI transfection reagents, where Turbofect is clearly the best.
Transfection itself is not the problem in this case. The question is why does transformation of a fully transfectable plasmid renders it non-transfectable?

Thanks for your comments!!



#126755 cells dont attach hard enough to 96 well

Posted by Gangwolf on 09 January 2012 - 07:01 AM in Cell Biology

If your cells is not dependent on specific growth factors and your assay is not too sensitive, you can even try skipping the PBS washing step. Just aspirate you medium and directly add fresh warm medium. It might help tilting your plate and gently add your medium to the wall of the well and then gently tilt back the plate to submerge your cells.



#126750 Transfection problem after transformation!

Posted by Gangwolf on 09 January 2012 - 06:32 AM in Cell Biology

Hi all!
I am trying to amplify a plasmid (P3XFLAG-CMV-14 plus non toxic insert) through backterial transformation. The plasmid that I am using works great for transfection in HEK293T cells, however, after transformation and DNA extraction, the HEK cells don't express the protein at all (or at very very low levels).

I have sequenced my insert and the CMV promoter and they are fully intact.

I have linearized my construct and it migrates at the same rate as the transfectable one.

I don't believe endotoxin is a problem, since I've tried using DNA extraction kits with and without endotoxin removal buffer. Plus http://www.ncbi.nlm....pubmed/10997275

I have tried different chemical competent cells (DH5-alpha, JMH109 & XL10 Gold) without success.

I appreciate any inputs!! Posted Image



#126483 difficulty in passaging HK-2 cells

Posted by Gangwolf on 05 January 2012 - 02:59 AM in Cell Biology

Try wash with PBS containing EDTA.



#126421 freezing adherant cells

Posted by Gangwolf on 04 January 2012 - 07:36 AM in Protein and Proteomics

No! Trypsin should not have any enzymatic activity at freezing temperatures. However, wash away the trypsin prior to seeding your frozen cells.



#126419 How to kill HRP ?

Posted by Gangwolf on 04 January 2012 - 07:33 AM in Protein and Proteomics

Or try incubating your blot in 0.4M NaOH for 20 min in room temp. on a shaker.



#125884 Black moving particles in the cell Culture

Posted by Gangwolf on 22 December 2011 - 02:29 AM in Tissue and Cell Culture

Take the supernatant containing the black dots and "culture" this in a cell culture incubator separately from your eukaryotic cells. If these dots are proliferating then I would say it is bacterial contamination.
Upon any bacterial contamination, discard your contaminated cells as they would give you inconsistant experimental results. In addition, contaminated cells might be very difficult to transfect.

Good luck



#125450 WT BRAF/NRAS mouse Melanoma cells

Posted by Gangwolf on 14 December 2011 - 08:58 AM in Tissue and Cell Culture

Don't know if you can open those pages. But anyway, Google can help. Those are human cell lines, though.
http://www.nature.co...ml#figure-title
http://www.nature.co...ml#figure-title
According to http://mcr.aacrjourn...entary_Data.pdf, also mouse B16-F10 are BRAF and NRas wildtype.

Thanks a lot!!!
I did the mistake of assuming that all the B16 sub-lines are BRAF mutated



#125417 WT BRAF/NRAS mouse Melanoma cells

Posted by Gangwolf on 13 December 2011 - 11:57 PM in Tissue and Cell Culture

Hi All!
I'm looking for mouse melanoma cells with wild type BRAF and NRAS. Alternatively, human melanoma with wild type BRAF and NRAS but has negative c-KIT expression.
I would really appreciate if anyone could point me to the right direction!

Wolf



#125416 HEK 293 cells Detachement

Posted by Gangwolf on 13 December 2011 - 11:51 PM in Tissue and Cell Culture

Use ExGen (Pierce) or Turbofect in vitro transfection reagent (Fermentas) for the HEK293 cells. These transfection reagents can be added to medium containing both FBS and antibiotics. I use both of these reagents regularly in the HEK293 cells with very good transfection efficiency. Turbofect is slightly better but it is being replaced by ExGen and might not be readily available.
If you'd like a protocol, just let me know!



#119446 protein expression vector..

Posted by Gangwolf on 12 September 2011 - 12:49 AM in General Lab Techniques

If you are not interested in eukaryotic post-translational modification of your protein, then use this system http://www.promega.c...slation-system/



#119310 Why do cells need to be washed in PBS before biotinylation?

Posted by Gangwolf on 09 September 2011 - 08:47 AM in General Lab Techniques

Probably to get rid of the serum proteins in your cell culture medium.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.