cell culture western blot - (Oct/19/2006 )
I want to do some western blot analysis on my cells, but I dont know how can I do this. Can I freeze the cells and at another time use RIPA buffer and the other experiments related to western blot?
How and when can I determine the amount of proteins?
Thanks in advance
I think there´s no problem about freezing and used them later. I have some samples freezed waiting to be run on a WB.
I thinks you can determine the amount of proteins by doing a typical Bradford assay or similar. I normally prepare at 1 mg/mL each sample, and then I add Laemmli buffer 1:1, which results in a protein concentration of 0,5 mg/mL.
hope this helps, although I´m not an expert.
I would suggest to freeze your lysate rather than your cells. Regarding the Bradford assay: it's not always possible to determine protein concentration using this assay because some reagents are not compatible with it in high concentration (like SDS and triton). Check the compatibility on the Biorad website; you could need to use another assay like BCA. When you load the gel, be careful you have more or less the same volume for all the samples if you want a good run (go up to the same volume with lysis buffer; 4-5ul difference is fine but 10ul is a bit too much for example).
Now I have a question: if you measure protein concentration and you load the same amount in each lane, do you need a loading control? I suppose it´s always better if you reprobe and check with beta-actin or the appropiate one, but what about the referees? Do they often ask for a loading control? thanks
I cannot tell you if they often ask because, as a matter of fact, I always probe with anti-actin or anti-nucleolin etc, even if I quantify proteins first therefore referees never asked me to. If I were to be a referee, I would ask because people can say a lot of things but, especially if they are showing something quantitative, they aren't always true. With a good loading control, I can believe the data. But even in that case, I would do a short exposure in which you can really see the difference among bands...
[quote name='dnafactory' date='Oct 19 2006, 12:00 PM' post='73684']
[quote name='pumuki' post='73681' date='Oct 19 2006, 10:53 AM']
Now I have a question: if you measure protein concentration and you load the same amount in each lane, do you need a loading control? I suppose it´s always better if you reprobe and check with beta-actin or the appropiate one, but what about the referees? Do they often ask for a loading control? thanks
[/quote]
Thanks dnafactory. My problem, as I posted in other topic some time ago, is that some of my WB are with a sample of human serum, and nobody can tell me about a good loading control for it. Only some suggestions about albumin, although I still haven´t tried it. 
[quote name='pumuki' date='Oct 19 2006, 12:11 PM' post='73688']
[quote name='dnafactory' date='Oct 19 2006, 12:00 PM' post='73684']
[quote name='pumuki' post='73681' date='Oct 19 2006, 10:53 AM']
Now I have a question: if you measure protein concentration and you load the same amount in each lane, do you need a loading control? I suppose it´s always better if you reprobe and check with beta-actin or the appropiate one, but what about the referees? Do they often ask for a loading control? thanks
[/quote]
Thanks dnafactory. My problem, as I posted in other topic some time ago, is that some of my WB are with a sample of human serum, and nobody can tell me about a good loading control for it. Only some suggestions about albumin, although I still haven´t tried it.
[/quote]
I confirm not to freeze cells but lysats for Wb; total protein amount to load Wb is only vague for specific analysis of certain proteins;
Do you think of albumin as loading control? If you work with blood plasm, ok, if you work with cells you only detect albumin as a rest of medium (if it contained serum) or if you added it to your sample, but this an after cell-lysis procedure and not acceptable to show equal loading; if you overexpress a protein or several, and in your Wb your are interested in this protein, you can stain it with Ponceau S or CBB; in this case, if you can reliably address a certain protein, you may use protein stain as loading control;
Do you think of albumin as loading control? If you work with blood plasm, ok, if you work with cells you only detect albumin as a rest of medium (if it contained serum) or if you added it to your sample, but this an after cell-lysis procedure and not acceptable to show equal loading; if you overexpress a protein or several, and in your Wb your are interested in this protein, you can stain it with Ponceau S or CBB; in this case, if you can reliably address a certain protein, you may use protein stain as loading control;
[/quote]
I worked with human blood serum, that´s why I´m thinking about albumin as a loading control.
then it is absolute ok to use albumin as an internal reference; you should simply detect albumin even with poor staining Ponceau S, scan it, destain and start your immunoblot; you will not detect appreciable amounts of beta-actin or tubulin (which will be predominantly from lysed blood cells, and are no reference for serum)
Ok, thanks a lot kosmodrom.
Only one detail: when I do the Ponceau staining (which stains all proteins, isn´t it?) it´s supposed that I will recognize albumin, apart from the molecular weight, because of the higher amount in comparison with the other proteins? 
PD: sorry saya for interfering a bit your thread 