I understand it is not your question but I have another question for you. What type of gel are you using. The gels I use have a loading capacity of 20 ug. These are Nupage Bis tris precast gels. So I'm really surprised you are loading 50ug. The amount sounds too much for any get! Just a suggestion to look into it! Also our wells also only need 20 uL because like Kenneth pointed out you don't want overflow!!

BTW Kenneth is correct it is 50ug/20uL!!! GOOD LUCK!

I'm a western queen considering i'm in a proteomics lab and its all I do. :P

If you're adding 50ug of protein and it's in a 20uL sample, you've loaded a concentration of 2.5ug/uL (50ug/20uL). That being said, I've never seen people refer to the concentration of a protein loaded into a SDS-PAGE - the important part is how much you load. So in this case, you should be fine reporting that you've loaded 50ug of protein per well. The only time I'd worry about volume is if you are unsure if that volume will fit in the wells without over flowing it.

Hope that helps!

I understand it is not your question but I have another question for you. What type of gel are you using. The gels I use have a loading capacity of 20 ug. These are Nupage Bis tris precast gels. So I'm really surprised you are loading 50ug. The amount sounds too much for any get! Just a suggestion to look into it! Also our wells also only need 20 uL because like Kenneth pointed out you don't want overflow!!

BTW Kenneth is correct it is 50ug/20uL!!! GOOD LUCK!

I'm a western queen considering i'm in a proteomics lab and its all I do. :P

To clear your doubt, if you measured your protein as x µg/ul and you are making the total amount as 50µg for your blotting in 20µl, this says it is 50µg in your whole 20µl. The only thing is concentration (x µg/µl) is diluted in 20µl but the final amount stays the same! (as Kenneth suggested).

To add some note, the amount seems bit higher for me (50µg), normally people use 10-20µg whole cell lysate for blotting. (you must increase if your protein of interest is less abundant!).

In contrary to Ariel, there is no gel with a protein capacity in µg! (you can theoretically load mg!, if you have such a high concentrated sample) I guess, she tried to mention as "µl". And I have no problem on loading 35µl volume in our biorad self-made mini gels. So this may be true only for the above said precast gels, doesnt mean that every gels have to be the same ;)

Thanks to all of you for your valuable comments, I am now assure that volume doesn't matter unless it doesn't overflow.

Hi Azaz,

I disagree with Ananda and partially agree with Ariel. I do not think the amount of protein you are loading is too much, it is probably at the higher end of what should be loaded. I am sure that if you go a supplier's website they specify the recommended amount of protein per well. If there is too much protein, it clogs up the pores, affecting run.

Hi Aniko,

I mentioned it as "Theoretically", there is no limitations gonna prevent from loading on the well, but it is our knowledge should guide us to decide the amount what we require for the need (western/coomassie staining). I agree the fact that it wont run properly/forms smear when too much amount is loaded, but it doesnt mean that you can not load it! :)

Hi Azaz,

You had a lot of useful suggestions already. I think I can post one additional comment. The amount of protein you load onto the gel is dependant on the type of the sample you have. For instance you can load 20- 40 μg of total protein from cell lysate, and much less 10 -100 ng of purified protein.

In fact if you want to do WB the amount you should load is also dependent on your antibody and the presumable concentration of your protein of interest in the lysate (if you are using cellular lysates). Naturally if you have good ab's and a lot of protein of interest in your lysate you can take much less on the track (10-15μg). But anyway it is better not to overload the gel, because the migration of proteins would be altered. You will have fused bands, shrink bands, wrong MW estimation and eventually bad pictures. Finally if you are making blot after PAGE you can stain your membrane with Ponceau S to verify that everything went fine.

Hope it would help.

Good luck.

Alexey

Dear Laia,

How many times concentrated sample buffer you use?

Be aware, that the detection limit in western blots depends very much on the affinity of your antibody on one site and the concentration of your antigen in your protein mixture on the other site. So you need a checker board titration to hit the optimal concentration of both for antigen / protein load on your gel and the concentration of your detecting antibody.

I would just like to know, loading a cell lysate on gel is a matter of amount of protein or concentration of protein? what matters here

Thanks Nigel... do you have a protocol or can roughly explain how you measured total protein concentration via nanodrop

Hello,

i have a question?

How to determine that the amount of loading protein is in dilute form or concentrated form???

thanks in advance...............

Hi Azaz, not sure if it's a little late in answering your question but normally, my lab uses BCA or Bradford Assay to measure total protein concentration. Preferably Bradford for me as it has fewer steps and thus, faster. Personally, I am very particular so I avoid using Nanodrop. It requires calibration and over time, it deviates giving inaccurate results, depending on the lab. So after determining the protein concentration via Bradford or BCA, you decide and determine the volume of the protein sample you want to use for Western. To give you a rough idea, I use a 32-ul loading volume for my samples - 20.8 ul sample, 8 ul NuPAGE LDS sample buffer (4X) and 3.2 ul NuPage reducing agent (10X).

I'm loading 50ug of protein irrespective of equal final volume per well. Is this correct or I have to equalize the volume also?

If you load a NuPage 3-8% Tris Acetate gel with more than 100 ug protein you will just waste your gel, protein and time. It will not run properly. The bands will be totally weird. This is not theoretical but based on experience. See the attached gel image with different ug of protein loading.

Dear Ariel Burn, I have a query regarding Azaz Ahmad question.

• "Either sample volume or sample concentration" which one should I consider first? Or we need to consider equally?
• For example, consider I will load 3 samples along with 2x sample buffer for gel electrophoresis. After bradford assay, I got following protein concentration: Sample-A: 5 µg/ µL; Sample-B: 7 µg/ µL; and Sample-C: 9 µg/ µL. And, I have to load 30 µg protein concentration in each sample. So, what will my final loading volume ?

Based on the protein concentrations, :

Sample A : 12uL + 8mL PBS + 20uL sample buffer (60 ug in 40uL)

Sample B: 8.6uL + 11.4uL PBS + 20 uL sample buffer (60 ug in 40uL)

Sample C : 6.7uL + 13.3 uL PBS + 20 uL sample buffer (60 ug in 40uL)

Then you can load 20uL per lane. Each lane will contain 30ug