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Irene G's Content

There have been 11 items by Irene G (Search limited from 15-November 18)


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#124714 Phospho-protein detection

Posted by Irene G on 02 December 2011 - 07:21 AM in SDS-PAGE and Western Blotting

We don't have any problems using this lysis buffer:
  • 150 mM NaCl
  • 50 mM Tris-HCl pH 7.5
  • 1% Triton X-100
  • 1% NP-40
  • 30 mM NaF
  • 5 mM EDTA pH 8.0
  • 1 mM DTT
  • 0.1 % SDS
  • H2O
You have to add just before using it:
  • 1 mM Na3VO4
  • 2 mM PMSF
  • 10 µg/ml aprotinin
  • 10 µg/ml leupeptin
  • 10 µg/ml pepstatin
Regards
Irene



#122378 intracellular cytokine staining

Posted by Irene G on 26 October 2011 - 05:49 AM in Flow Cytometry

You must write your protocol, if not, it is difficult to help you



#122376 Re-using membranes with ghost bands

Posted by Irene G on 26 October 2011 - 05:42 AM in SDS-PAGE and Western Blotting

Thank you, I will make a new gel!



#122375 Low Molecular Weight Housekeeping Gene for Loading Control?

Posted by Irene G on 26 October 2011 - 05:40 AM in SDS-PAGE and Western Blotting

Tubulin has a band of aprox 50 KDa, maybe you can use it, or GAPDH if you change the secondary antibody (if your first Ab is made in rabbit, you can buy a GAPDH Ab in mouse)
Hope it helps you
Irene



#122361 longer running of proteins in SDS PAGE!

Posted by Irene G on 26 October 2011 - 02:47 AM in SDS-PAGE and Western Blotting

Which % of acrylamyde are you using? You can try to change this in order to separate the three bands and not loosing one of them.



#122359 Re-using membranes with ghost bands

Posted by Irene G on 26 October 2011 - 02:40 AM in SDS-PAGE and Western Blotting

Hi!
My question is if I can reblot a membrane with ghost bands again with the same antibody but less concentrate. Is this possible or I have to repeat the gel?
Thanks
Irene

Attached Thumbnails

  • blot.jpg



#111065 Keratinocyte Primary Culture

Posted by Irene G on 27 May 2011 - 01:31 AM in Tissue and Cell Culture

Hi!
I don't culture keratinocytes but I think you might ask Lonza for the optimal culture conditions for this cells.
Good luck
Irene



#97133 Monocyte Isolation from Whole Blood via Gradient Centrifugation

Posted by Irene G on 11 January 2011 - 03:01 AM in Tissue and Cell Culture

I use Histopaque 1077 from sigma to obtain mononuclear cells from a buffy and then I do positive selection with CD14 (miltenyi) I tried percoll before and obtain few cells and the same with adherence.
Good luck



#97132 Fungal Contamination?

Posted by Irene G on 11 January 2011 - 02:52 AM in Tissue and Cell Culture

I'm agree with you, it has to be fungal contamination, throw away the contaminated flasks and pray for the others stay clear. Spores are everywhere and are very difficult to remove.
Good luck



#97131 establishment of primary cell line from biopsy

Posted by Irene G on 11 January 2011 - 02:47 AM in Tissue and Cell Culture

Hi everyone,

I'm trying to establish primary cell lines from bladder cancer samples. I keep trying, but after a while I still cant have any growth at all!

I've been using this protocol where I cut the sample into little pieces, seed it and then add some F-12 medium + non essencial a.a., dextrose, insulin, glutamin, P+S as antibiotics and FBS.

Cant really understand where is the problem, no article describes this problem or how to fix it, im really getting worried.

Thank you in advance :)
Happy 2011 everyone

Nya


Try disgregation with collagenase, maybe it could help!



#97130 Help! How to prepare 1 x 10^6 cell

Posted by Irene G on 11 January 2011 - 02:45 AM in Tissue and Cell Culture

Hi!
I hope I could help you:
First rinse cells with 5ml PBS, after that add 1-2 ml of Trypsin-EDTA, put them in the incubator, after 2-3 min look them (microscope) and observe if they detach, if that doesn't happen, leave them 1-2 min plus. Add PBS (8-10 ml) and collect all (liquid+cells) into 15 ml falcon tubes. Centrifuge for 5 minutes at 1500 rpm and then discard supernatant and resuspend cells in 1 ml medium. Count the cells in a hemocytometer and add the medium necessary for having 1x10^6 cell/ml
Good luck




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