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mikew's Content

There have been 23 items by mikew (Search limited from 23-October 19)


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#68758 Number of proteins expressed in cell

Posted by mikew on 29 April 2010 - 02:18 PM in Protein and Proteomics

Hi,

Does anyone know how many different proteins are expressed in a mammalian cell?
I know there is supposed to be between 10 - 20 thousand but can anyone give me a reference for this?
Has there been a proteomic study done to answer this question?



#40597 HA-TAG

Posted by mikew on 21 October 2009 - 10:49 AM in Protein and Proteomics

Hi,
I have HA tagged my protein. My sequencing reaction looks good. Everything should be in frame.
I have added a single HA tag.
The problem is that my protein is expressed (by Western) but I cannot detect it with an HA tag antibody (I use a 3x HA tag for control).
Any ideas?



#37220 help!! immunoprecipitation

Posted by mikew on 22 September 2009 - 11:05 AM in Protein and Proteomics

In addition to needing an input as stated above, what species are your antibodies?
If the Ip antibody is rabbit you should use a different species for the Western?
Do you have proper controls? Have you done the IP using a cell line that was previously published?



#36141 trouble to grow cells on top of coverslip

Posted by mikew on 11 September 2009 - 01:25 PM in Cell Biology

Some cells do not grow well on cover slips unless they are poly-lysine coated.
However, I normally submit the cover slips to UV light in the hood to sterilize and then seed.
I don't think you need to HCL wash the slips, this may change the charge of the cover lsips and lead to less binding.
I put the slip into a 24 well plate add media and THEN add the cells. This order seems to work well.
Also, using fresh media makes a difference.



#34518 nuclear extract -- HEPES pH?!

Posted by mikew on 26 August 2009 - 12:16 PM in Protein and Proteomics

Different protocols use different pH because that is what is on their bench at the time.
I always use pH 7.4. I don't think it matters as long as you are within the 7.4 -8 range.



#34517 Purifying a strange native protein

Posted by mikew on 26 August 2009 - 12:13 PM in Protein and Proteomics

My experience is that there is always a sizeable amount of protein in the flow through.
I always pH my buffer to 7.4.
Did you dialyze your sample? What is the NaCl concentration? Is it low enough for binding?
Also, run the column as slow as possible. I get much better binding the more patient I am.



#32247 help!! immunoprecipitation

Posted by mikew on 07 August 2009 - 01:28 PM in Protein and Proteomics

How large is your protein? Is it a similar size to the IgGs?
Is the protein expressed in the cell type you are using?
Have you used a cell line that was previously used successfully by other groups?



#31528 Gel shift (emsa) with 35S label?

Posted by mikew on 03 August 2009 - 09:12 AM in Protein and Proteomics

The important thing (VERY IMPT!)
is to do a negative control with rabbit lysate
that does not have your protein. Eg. 35S label another unrelated protein as a negative control.
The lysate will bind many many probes.
If you do not have a negative control, label the oligos with 32P and
use lysate as a negative and your translated protein as the unknown.
There are many many proteins in rabbit reticulocyte lysate that bind DNA,
it is very easy to get artifacts.



#31232 Variable expression of antigen in various subcellular locations - Any ideas why?

Posted by mikew on 31 July 2009 - 11:44 AM in Cell Biology

Gonna go out on a limb here and ask if you see different pattern of staining for other
cell surface proteins?
The reason I ask is that if your cells are intact, they are covered by a membrane. This protein covers the membrane so it should look like its present everywhere.
Picture a basketball flattened and you are staining for something on the surface of the basketball. It will look like it is nuclear and cytoplasmic, but it is merely on the surface of the cell.
Otherwise, maybe the antibody is crappy.



#31231 Gel shift (emsa) with 35S label?

Posted by mikew on 31 July 2009 - 11:38 AM in Protein and Proteomics

The TNT kit comes with a protocol for S35 labeling.
However, the signal is weaker than 32P labeling of your DNA probe.



#29865 Would transfected plasmids bind histones in HeLa cells?

Posted by mikew on 21 July 2009 - 12:24 PM in DNA Methylation and Epigenetics

Histones integrate into plasmid DNA in the nucleus.
But apparently the placement isn't the same as the
endogenous locus. But they're there.



#29864 2X sample buffer vs. 6X

Posted by mikew on 21 July 2009 - 12:20 PM in Protein and Proteomics

The difference is 4x :D.



#29863 Extraction of protein from sample buffer (+DTT)

Posted by mikew on 21 July 2009 - 12:19 PM in Protein and Proteomics

Run on gel and silver stain.
Is this a test question?



#26769 How to store membrane with primary antibody?

Posted by mikew on 15 June 2009 - 10:46 AM in Protein and Proteomics

It should be fine.
I would probably reincubate for 1 hr with the primary.
Could you ask another lab for the secondary?



#26554 Supershift Problems

Posted by mikew on 11 June 2009 - 02:34 PM in Protein and Proteomics

Hi,

#1. Your purified protein binds the probe that comes with the kit (whatever that is).
This indicates that your protein will bind any probe non-specifically.
#2. The results of the binding with your probe and the mutated probe looks reasonable.
If you leave out lane 10 the lanes from 4-9 would look logical. Your protein shifts with the antibody and the mutated probe doesn't bind.
However, you do have lane 10. So, it appears that the His-Antibody is complexing with some protein in your mix
to form a stable protein-DNA complex. This complex seems to form with any probe.
It would be a good idea to include BSA and detergent (NP-40 or Chaps) in your EMSA mix.
This may make the binding of the protein somewhat more specific.



#25979 Co-IP hassle

Posted by mikew on 04 June 2009 - 09:57 AM in Protein and Proteomics

Okay, so your myc-tagged protein is IPing well if I understand correctly and
the efficiency of the myc-IP is high judging by your inputs.

Is it possible that you are not IPing enough material? How much are you IPing?
Maybe you need to increase the amount to 2 mgs or so.



#25722 Co-IP problems with band sizes

Posted by mikew on 01 June 2009 - 02:03 PM in Protein and Proteomics

Photo?
Also, the salt concentration and detergents in your samples can affect running mobility.
Try to ensure that buffers in input and IP are consistent.



#25721 Co-IP hassle

Posted by mikew on 01 June 2009 - 01:57 PM in Protein and Proteomics

Is it possible that your protein does not interact with the target protein tin the cell line you are using?
Maybe you could use the cells previously published as a positive control.
I think Isek makes some very good points as well.



#24039 Co-IP problem

Posted by mikew on 11 May 2009 - 12:09 PM in Protein and Proteomics

Hi,

#1. Don't do westerns of an IP with the same species antibody. This leads to artifacts and non-specific bands.
#2. Your heavy chain migrated faster in the IP lane probably due to a different amount of antibody being in that lane. Signal is weaker in control lane due to different amount of antibody.
#3. As for the smiley face, I don't really know, make new buffers and maybe run your gel more slowly.
Smiley bands usually result from heat.



#23733 anti-phosphotyrosine blot on 2mo old samples?

Posted by mikew on 07 May 2009 - 03:14 PM in SDS-PAGE and Western Blotting

Personally, I've never reused samples.
I run IP samples or whole cell lysates on gel and then proble for Phosphorylation.
The quality of the extract for this type of probing changes over time. Proteins degrade, phosphorylation is lost etc.



#23732 In Vitro transcription/Translation

Posted by mikew on 07 May 2009 - 03:09 PM in Protein and Proteomics

How did you analyze your bands?
Western? S35 labeling?
This makes a huge difference.



#23390 phosphotyrosine blot

Posted by mikew on 04 May 2009 - 11:21 AM in Protein and Proteomics

Hi,

Yep, I bet the problem is that you are using a pan-phosphotyrosine antibody.
If you use a phospho-specific one I bet your experiments will work.
Glad to hear you have a control for your LPS activity, that always a good idea.
I have used pan-phopho-tyrosine, serine and acetylation antibodies and haven't seen changes on a global scale.



#23159 phosphotyrosine blot

Posted by mikew on 30 April 2009 - 01:55 PM in Protein and Proteomics

Hi Max,

I have no experience with dendritic cells.
However, I have treated various other cell
types with various stimuli that is supposed
to induce phosphorylation and have never seen
a change on blots from whole cell lysates.
I only see changes when I immunoprecipitate
specific proteins and look for specific changes.
The reason for this is that in a typical cell 66% of the proteins are already phosphorylated, if you induce with
LPS or whatever, and this induces hyperphosphorylation of 100 proteins this is still a small fraction of the total.
Also, you may not notice hyper phosphorylayion versus basal phosphorylation on a whole cell lysate basis.
Do you have a reference for where people have seen this sort of change?
Also, check to make sure your LPS treatment is working.
For example, is NF-kB activated or some other control?




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