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!

drwho's Content

There have been 23 items by drwho (Search limited from 10-August 19)


Sort by                Order  

#143751 Controls for RT-PCR reactions

Posted by drwho on 19 October 2012 - 10:28 AM in PCR, RT-PCR and Real-Time PCR

Hi

Just to clarify beta-actin and GAPDH are not positive controls! A positive control would be for your gene of interest we have used plasmids containing our gene of interest to perform absolute quantification, this is considered the gold standard but is time consuming.

The genes you are talking about, GAPDH and beta actin are often used as house keeping genes ie they allow for normalisation but ARE NOT POSITIVE CONTROLS! You need to understand this, otherwise your experimental design is flawed!

To establish which housekeeper to use you should look a the expression of these genes during the course of your experiment, if they change they are unsuitable if they remain constant they will suffice.



#143725 Could a change of FBS affect outcome of a cell proliferation assay?

Posted by drwho on 19 October 2012 - 07:06 AM in Tissue and Cell Culture

Yes FBS will make a difference for your assay. The simple answer is that it comes from different animals you will always have variation in growth factors and various other things between these animals they may different ages the list could go on forever.

You should ideally batch test very set of serum you get in your key assays to see if it changes.

Most companies will send out a free sample for you to do these tests as long as you buy a large quantity. Also they will keep you batches and send them out to you as required if you do not have space to store large quantities. We tend to order 100 500ml bottles in a batch.

Hope this is helpfull



#143668 How do they make passage limited cells?

Posted by drwho on 18 October 2012 - 10:27 AM in Tissue and Cell Culture

one way is to irradiate the cells, which stops cell division. We have had such cells from perkin elmer eg http://www.perkinelm...12F_M1F-A12.pdf



#143634 Lentivirus kills 293T?

Posted by drwho on 18 October 2012 - 07:40 AM in Cell Biology

This may be a typo you said you are seeing red fluorescence? This may be really simple but are you using the right filters on your microscope. If you are using GFP (Green fluroescent protein) you should not be seeing red as you mentioned above.

GFP excites at ~395nm and emits at 509 just double check this



#142821 Effect of GTP on ligand binding.. confused

Posted by drwho on 04 October 2012 - 01:00 PM in Pharmacology and Pharmaceutics

Just clarify a few things GTP does not bind to the receptor but to the G-protein.

In terms of GTP lower receptor affinity, this could be in the case in membrane binding preps. This is because receptors can exists in two states a low affinity sate ® this is when the receptor is not coupled to the G-protein. When the receptor is coupled to its G-protein it is in a high affinity sate (R*), this is the inactive G-protein so to say when it is in association with GDP. So when you add GTP it causes the G-proteins to become activated so they are not associated with its receptor and so lowering affinity entering the R state.

There could be many explanations for what you are observing, do your see changes in the efficacy (Emax)?

I suggest reading this excellent paper on receptor theory

http://www.sciencedi...22519399909888#

Hope this helps



#142747 The 3rd reviewer - video

Posted by drwho on 03 October 2012 - 01:50 PM in Journal Discussion - How to Get Papers Published

love this, scarily accurate



#142746 Where are you from?

Posted by drwho on 03 October 2012 - 01:43 PM in Chit Chat

originally Wales in the UK



#142727 Reducing cell adhesion

Posted by drwho on 03 October 2012 - 11:56 AM in Tissue and Cell Culture

One way of doing this may be to use calcium free PBS this should remove any non strongly adherent cells. I have some cells where I use PBS instead to trypsin to detach them from my flask. Might be worth a try is nice and simple!

Hope this is useful



#142725 Best ECL detection reagents

Posted by drwho on 03 October 2012 - 11:47 AM in Molecular Biology

In my experience the ECL you is very dependent on your on you protein of interest, I work on proteins which are expressed at very low levels under native conditions so we use this http://www.piercenet...?fldID=01041102 because it allows us to do long exposures and is very sensitive. However if you use it on proteins which are expressed at moderate to high levels it will give you very strong bands unless you do very short exposures

This may draw great deal of derision but for westerns of well expressed proteins I use a Santa Cruz ECL (http://www.scbt.com/...ol-reagent.html) I have no particular reasons expect that it has always worked for me and is what I have always used and it is cheap!

But recently we have started to use quantum dot based reveals with a great deal of success due to the strength of the signal we can load less protein and so produce much cleaner blots than we ever previously were able to obtain and multiplexing is possible. However this does require addition pieces of equipment but in its most basic form can be done on a transilluminator.

Hope this helps



#142560 How to concentrate a small peptide < 2 kDa?

Posted by drwho on 01 October 2012 - 04:11 AM in Biochemistry

Hi I have tried this dialysis method with good results:

http://www.piercenet...?fldID=04010160

But it goes down to 2kda so may not work out for you, their may be others manufactures out there who may go smaller.

You could also try freeze drying you protein then re-suspending it any volume you want, there is literature out there which says this can alter your protein and so affect function but I never had this issue.

Hope this helps



#142555 Lentivirus kills 293T?

Posted by drwho on 01 October 2012 - 02:08 AM in Cell Biology

No sorry I have not done anything like that, I have only done rather simple shRNA plasmid packaging. This was a very long time ago we tend to buy particles in now!

Could you go into a bit more detail, about exactly what you have done and I will see if I can spot any issues. But I can not promise anything :)



#142485 IGF1 stimulation of THP1 cells

Posted by drwho on 29 September 2012 - 02:01 AM in Tissue and Cell Culture

I would recommend you produce a dose response curve for this type of work, and determine your own EC50 in your hands and in your specific cell type. This should be easy as you have a dose range I would do 7 doses, it will be essential to the the top and bottom of your sigmoid curve.

This might seem a bit repetitive of published work but in my experience is essential (particularly if you want a good publication) as peptide ligands can be very tricky to work with as they can "stick" to plastics tips etc and each lab uses different consumables so variations are common.

In terms of read out are you looking for a secreted marker? But if you are looking for receptor activation I have done work with a phospho-IGf1R antibody: http://www.cellsigna...ducts/6113.html which is quite good.

This will give you a absolute measure of activity as this is the first signalling event in the IGF-1 cascade.


Hope this is usefull



#142417 RNA isolation from small numbers of cells

Posted by drwho on 28 September 2012 - 05:07 AM in Molecular Biology

Thanks for the reply Trof

qPCR will be my final application. I think I will proceed with the PCR rather than do any further clean up steps, as you are right I am likely to loose more sample.

The cells I am looking at a specific sub-population of cells, but I also have larger amounts of the whole population of cells which have been prepared in the same way but have good quality and quantity of RNA. I was thinking of comparing the housekeeping genes between the populations, this should tell me if the contamination has affected my reactions and whether my data is any good. I would be grateful for you thoughts on this approach?

Thanks in advance



#142412 Lentivirus kills 293T?

Posted by drwho on 28 September 2012 - 02:46 AM in Cell Biology

just a general comment, I do not have specific experience with viruses in HEK cells. However all viruses will be toxic to cells if there are enough of them! Do you know the concentration you viruses so you can check your MOI (multiple of infection). If your MOI is very high you will get non specific death due to large numbers of viruses clogging up the cells.

If you do not know the virus number, try doing various dilutions of your stock virus and see if your cells are more happy.

Hope this helps



#142410 Puromycin - Stable

Posted by drwho on 28 September 2012 - 02:17 AM in Tissue and Cell Culture

what I have done in the past is 24/48 hours prior to you needing your media containing your chemokine remove your antibiotic containing media and replace with your normal media (with no antibiotic). Collect that media (24/48 hourse later) and use as required, then replace with your selection media. If the cells are stable a few days out of section should not affect them too much



#142377 requesting your assistance for IC50

Posted by drwho on 27 September 2012 - 01:32 PM in Cell Biology

IC50 is a product of a curve so you need to make sure your curve is good. So this mean you need to have enough points on your curve to make it a good fit (if you do not understand what I mean by this you need to understand the mathematics of curves) I would never use less than 7 points. To clarify what I mean by 7 points is 7 doses of your drug. These really should be log doses as show in the link in the above post.

What you need is the top and the bottom of your curve if you can not find these your IC50 will not mean anything.



#142374 research ideas

Posted by drwho on 27 September 2012 - 01:05 PM in Research Idea, Design and Collaboration

I agree with the above post. I think you are on the right track.

I would suggest you ask your self why augmentin is used think out how its composition is related to its use. One of the best lessons I was ever taught was "how does structure relate to functions" if you keep this in mind you will do very well. Always ask this question



#142373 study of the role of a protein in a lesion

Posted by drwho on 27 September 2012 - 12:56 PM in Research Idea, Design and Collaboration

my first thoughts about study anything like this would be to ask what tools you have:

Anti-bodies
siRNA/ KO animals
Small molecules inhibitors

Quite often these are not available in new targets, my next thoughts would be on what is common to other systems so look at common signalling systems and work backwards. I agree with the above comments in the expression in pathology and physiology I would add to this by (given your example is cancer) look for mutations and then asses function etc.

A bit of basic advice is know the literature including meeting abstracts patents etc think out side of pubmed!



#142372 New and creative ways to teach science!

Posted by drwho on 27 September 2012 - 12:43 PM in General Biology Discussion

I am a big believer in getting people/kids involved, I am a UK based scientist and I regularly go out into schools. The best responses I get are in my practical demonstrations the best example I can give is how bone is a composite material ie protein (collagen) and mineral (calcium). The way I demonstrate this is using concrete platform with and without steel bars in (bear with me) I get the kids to jump use hammers and any thing else they can find to try and break these materials, gets a bit mad and a health and safety nightmare. One set they can break easily the other hard as they might I have only had one class manage to break the reinforced concrete (very angry children :) ).

But shows in a practical way how different materials are stronger than other and how this relates to how your skeleton and many other areas of science.

As a research scientist, I get to go into schools on a infrequent basis and get to do cool "showy" things, but I have so much respect for the teachers who can make their subjects interesting on a daily basis.

Hope this helps



#142371 RNA isolation from small numbers of cells

Posted by drwho on 27 September 2012 - 12:16 PM in Molecular Biology

Thanks for the reply. If it were any other sample I would just give it ago but given the scarcity of my sample I am canvassing for opinions!

Do you have experience of PCRs using RNA with low 260/230 levels? I would be more conformable if all the values were the same between the patient samples ie equal error. But they are different enough to make me concerned.

Cheers



#142370 Dilution factor 50 ul in 2mls

Posted by drwho on 27 September 2012 - 12:04 PM in General Biology Discussion

Small addition to the above post, make sure everything is in the same units. For example you state the dilutions as 100ul in 2ml it is much easier to think of as 100ul in 2000ul



#142364 Distinguish murine and human cells by surface staining?

Posted by drwho on 27 September 2012 - 11:41 AM in Tissue and Cell Culture

Hi depending on your cell type, we have reasonable success with this:

http://www.stemcells...Catalog/STEM121

Hope it works foe your guys



#142360 RNA isolation from small numbers of cells

Posted by drwho on 27 September 2012 - 11:23 AM in Molecular Biology

Hi all

I hope you can help I have read many topics on this issue but thought I would ask for some help.

I am trying to isolate RNA from a small numbers of cells ~1000 (human samples). In my trial experiments using cell lines I managed to get usable quantities and quality RNA using Guanidinium thiocyanate-phenol-chloroform extraction (TRIZOL) with an additional ETOH sodium acetate precipitation, also tried a butanol concentration set with good results. I was able to repeat this in a number of differnt cells types.

However now I have tried it on the real samples I have good quantity and good 260/280 values however my 260//230 values are low ~1.3-1.5. I am at a bit of a loss about what to do. I will be unlikely to get more of these cells for a long time if at all.

Any advice on how I can improve my 260/230 values. I will want to use the RNA for standard real time PCR no arrays or anything like that.

Any suggestions more than welcome.




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

©1999-2013 Protocol Online, All rights reserved.