Hi Huaqing-
I would not give up on the immunofluorescence localization. Each antibody has its own peculiarities, and often needs a specific set of fixation and staining protocols to work. I've seen the same antigen injected into two different rabbits produce polyclonal antibodies where antibodies from one rabbit only work well in PBS and the antibodies from the other rabbit only work well in Tris-buffered saline. Annoying. You will have to vary 3 parameters to explore the parameter space, going through a bunch of coverslips:
Axis 1 is fixation: Try (each of the following is a different method)
-ice cold methanol
-room temp 95% ethanol
-room temp 3% fomaldehyde in 20 mM phosphate buffer pH 6.5
-room temp 3% fomaldehyde in 20 mM phosphate buffer pH 6.5 10 min followed by 10 min at room temp 95% ethanol
-room temp 3% fomaldehyde/ 0.1% glutaraldehyde in 20 mM phosphate buffer pH 6.5 (remember to work with glutaraldehyde in the hood)
-anything else you can think of
all 10 minutes, followed by PBS/ 0.5% NP-40
Axis 2 is buffer: for all of the fixation methods above, try the antibody at 1:50 (if at all possible, have the antibody purified with Protein A beads to get fairly pure IgG, make sure this IgG fraction works for you on Western blots, some antibodies are mostly IgM), using each of the following buffers for the primary antibody incubation and the primary antibody washes (3 washes, 5 minutes each); then just use PBS/ Tween for the secondary antibody 1 hour room temp and 3 washes in PBS/Tween
-Phosphate buffered saline (PBS; 130 mM NaCl, 20 mM Phosphate pH7.2)
-PBS/ 0.05% Tween 20
-PBS with 1 mM MgCl2
PBS/ Mg/ 0.05% Tween 20
-Tris buffered saline (TBS: 130 mM NaCl, 20 mM Tris/HCl pH7.2)
-TBS/ 0.05% Tween 20
-TBS/ 1 mM MgCl2
-TBS/ Mg/ 0.05% Tween 20
-and each of the above 8 preceded by an antigen retrieval step (typically heat sample to 95 degrees C in citrate buffer pH 6; google this for details)
Axis 3 is stain temperature; try all of the above with the primary antibody incubation done in a humid box
-37 degrees for 1 hour
-Room temp for 1 hour
-4 degrees overnight
once you see some staining, you can then start dialing back the primary antibody concentration, and if there is a lot of background, start going harsher on the primary incubation and wash conditions - add .3 M NaCl, or .5 M NaCl, or high salt and 1% NP40, or (harshest) high salt/ 1%NP-40/ 0.1%SDS
good luck
Richard Gomer
Department of Biology
Texas A&M University
301 Old Main Drive
College Station, TX 77843-3474
979 458 5745
________________________________________
From: DICTY [[log in to unmask]] on behalf of Thierry Soldati [[log in to unmask]]
Sent: Sunday, June 6, 2021 1:30 AM
To: [log in to unmask]
Subject: Re: [DICTY] organelle fractionation
Dear Huaqing,
I fully agree with Rob.
There are methods to separate organelles, and if you wish, I can un-earth some protocoles we used for plasma membrane (gold colloid coating), endosomes and phagosomes (latex or paramagnetic iron loading). Mitochondria and nuclei are a standard. But sucrose gradients, for example never deliver “pure” fractions, but a series of overlapping peaks, and to do a good job, you will need a dozen antibodies against markers of the various compartments. A daunting task …except if you can good-guess where it could be (by analogy, or computing a prediction) and use the right protocol to confirm.
If you would describe better what you tried and what failed, I am convinced we can help you with other suggestions to localise your protein by microscopy.
- Do you have an antibody (or antibodies) to your protein? You could try the recombinant antibody facility from Pierre Cosson to obtain more.
- Did you try N-term or C-term tagging? Or even in an internal loop? You should try the ALFA tag, which again Pierre advertised some time ago. It truly is the best tag ever for Dicty.
- Did you try extrachromosomal vectors for expression? The pDM series and Peggy Paschke can surely give you the best advice on that.
- Did you try chromosomal tagging by knock-in? We have had very good success with C-term GFP tagging, but in case this “hurts” your protein, try the ALFA tag.
Good luck,
Thierry
On 5 Jun 2021, at 22:37, Rob Kay <[log in to unmask]<mailto:[log in to unmask]>> wrote:
For what it's worth, and based on my experience with rat liver homogenates
long ago, I would not even try fractionation!
It's worth doing a very basic fractionation into nuclear/mitochondrial,
endoplasmic reticulum and high speed sup fractions, but beyond that, I
think it is very hard to get definitive assignment to Golgi, contractile
vacuole, or vehicles of the endocytic pathway. The various sorts of
gradient you can use will give broad, overlapping fractions and if you are
looking at vesicles, you will have to work hard to keep them intact.
Better to improve your tagging or antibody......
Rob
Dear Dicty-colleagues,
We are trying to figure out the subcellular localization of a membrane
protein. Due to tagging and expression issues, we are not able to do this
by microscopy. However, we can detect the protein by Western blot. So we
are wondering if it is possible to determine the localization by
subcellular fractionation.
Does anyone have a good protocol for separating different organelles in
Dicty? Such as sucrose or percoll gradient?
Many thanks in advance,
Huaqing
_________________________________________
Prof. Dr. Huaqing Cai
Institute of Biophysics
Chinese Academy of Sciences
15 Datun Rd, 6223, Chaoyang District
Beijing
China
—
NEW OFFICE Room 344C
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Prof. Thierry Soldati
Department of Biochemistry
Faculty of Science
University of Geneva
30 quai Ernest Ansermet, Sciences II
CH-1211-Genève-4, Switzerland
Tel: +41-22-379-6496
email: [log in to unmask]<mailto:[log in to unmask]>
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