There can be many reasons:

Dilution of primary and secondary antibody

Type of membrane

Not enough Protease inhibitor and so on....

One I had the same problem. I just changed the non-fat dry milk powder. I bought it just at a supermarket and my proteins became visible. Worked better then the expensive once from Biochemical companies.

Have you already tried increasing the amount of sample load, concentration of antibody and reducing the washing in your modifications?

Stephen's suggestions are worth trying. Also you can try to use another chemiluminescence kit. We changed our chemiluminescence kits to BioRad lately and they are highly sensitive for the detection of faint bands

Initially make sure you have proteins transferred to your membrane, stain the membrane with ponceau buffer upon 10 min in the shaker you should be able to observe bands. If not you don't have proteins transferred. You mention your bands are weak...expose the film....more...time, make sure your antibody does not need to be diluted in BSA... and i agree with Atakan Ekiz about the BioRad kit it is very sensitive to detect weak bands

you can try 2 things first by protein extraction use 0.5% NP40 second u can increase the transfer time

Yes I think I had this problem especially when I was not using the protease inhibitor cocktail tablets

I would check the transfer with ponceau first. Your ECL solution you can check easily, just mix a drop of the solution, put a tip in you HRP antibody and then in the dark in the drop, it should glow immeadiatly. I would recommend protease inhibitors in all samples.

what kinf od anti-body did you use?mono or policlonal?

how long anti-body is prepared?

We always used GEHealthcare Amersham Hyperfilm ECL as chemiluminescence kits and we have great results!

I tried the modifications mentioned, I'm using the movie to Amersham Hyperfilm ECL revelation, and strong western Luminata (Millipore). Does changing the film for autoradiographic improve? Use of the protease sigma, and freeze at-20C. Quantification of protein was determined by Lowry method, since the Bradford interfere with RIPA.

Always the primary antibody. Try another one and good luck!

is it possible that you have not loaded enough sample? If your protein is not present like actin or tubulin , you could try to load more , even 70 ug of total proteins.

I agree with Irene, you need an internal control as tubulin and actin to know if you have or not a sufficient amount of your protein also.

ponceau red can detect proteins up to 200ng. Below this amount, you can also have a white membrane, but doesn't mean that you don't have proteins on it!

I agree, you should check for a housekeeping, such as GAPDH

I second the protein control but I also stain the gel after transfer to see how much protein was left behind. Wet transfer is better for low level of expressed protein. If you are using ECL there are new pico kits which are more sensitive then the old ECL.

Also try increasing your primary concentration as long as you do not get background it is a good way to go!

And your secondary antibody is alright? May be it could be old or not good enought to emit fluorescence

You can do dot blot to check whether primary and secondary Ab's are working fine. Sometimes there is a chance of protein blow through during transfer.If the results for house keeping gene are Ok then try increasing protein concentration or primary Ab .

There could be many causes. Rajesh Ramakrishnan has given good tips and I will add the following to that. Depending on the size of your protein you could be transfering for too long or too short of a time (larger proteins need longer to transfer), and you could be using too much or too little methanol in your transfer buffer. Also are you using PVDF or nitro-cellulose?

As many have suggested, you should look at the levels of your loading control like actin or tubulin, if they are also giving weak signals then it could be either your transfer or you are not loading enough protein. If you got a good signal for your loading controls then you probably need to increase the primary antibody concentration or incubation time, also try increasing exposure time with the chemiluminescence.

I hope this helps.

Dear Tiane,

If the background is oke and the bands are at right size then is the first antibody specific and you can increase the sensitivity at least 80 times compared to HRP using Alkaline phosphatase based chemulum substrates like Roche's CDP-star. ( Do not forget to use Levamisole during blocking step to inhibit endogenous alkaline phosphatase activity).

Higher load of protein may results in immidiate activation of signal in ECL based protocols prior the detection (you would observe very rapid decrease of the signal of specific band compared to noise background). In that case you can develope bands using chromogenic DAB instedd of ECL ( reducing sensitivity up to 10 times).

Good Luck

I use nitrocellulose membrane, which seems incredible to not get good results with memebrana PVDF. Use the Chimioluminescence Hyperfilm ECL, is the Hyperfilm ECL autoradiography is better?

Thanks for the suggestions!

Thanks Markus! The amount of protein I was used to 100ug. I'm guessing that the method of Lowry protein quantification is overestimating my sample. I read articles that were able to detect proteins of apoptosis with 7ug. That way I never get it. I'm thinking of dialyze the samples to quantify by Bradford. Your suggestion is interesting film. I tried to prove a common good but also Kodak's automated device was used in those hospitals. I use the manual form of revelation: developer - water - fixer. I'm trying to make a breakthrough with secondary antibody coupled to FITC. Reading CCD camera. The result so it was good.

There is something special to work with the PVDF membrane as well as place it in methanol for a few minutes and then put it in transfer buffer for 15 minutes? I did not get good results. The pounceau not stained bands and in revealing the membrane was all dark. I read that after the transfer the membrane would have to wait to dry, put in methanol and then again following, blocking ... I even tried doing this but I think it was worse. Can transfer the nitrocellulose membrane with PVDF?

On the PVDF membrane you need to wet with methanol for 30 seconds and place into your transfer buffer for 5 minutes, but after your transfer is complete do not let the PVDF membrane dry, as there protocol suggests. I immediately place the membrane in blocking solution (5% Milk) for 1 hour before adding my primary antibody. Also if your looking for film that is extremely sensitive and cheap, we use Denville's film called HyBlot CL, Great stuff. Finally I use Pierce chemilumenescent products West Pico or West Dura depending on the difficulty of seeing the protein. If I know a particular protein is difficult to see then I will use the Dura because of its increase in sensitivity detection.

You can also use a premixed substrate - Merck Luminata. There are 3 different substrates. We have used the Luminata Forte and it was much better than with normal ECL.

Hi, Here there can be two problems, one if proteins have not been transferred properly. So for that you need to use prestained markers if available. That will make sure that protein has been transferred properly. The second problem may lie in detection of your interest protein. For that you have to optimize the concentration of your primary Ab which you are using. You can also try DAB with metals like Nickle which are used as enhancer.

There can be many reasons:

Dilution of primary and secondary antibody

Type of membrane

Not enough Protease inhibitor and so on....

One I had the same problem. I just changed the non-fat dry milk powder. I bought it just at a supermarket and my proteins became visible. Worked better then the expensive once from Biochemical companies.

Great idea Andreas! I'll try ess change since my milk powder is old. The other suggestions are also tested.

Dear Tiane,

It is long shot but maybe worth trying: Instead of using commercial ECL, just try the following custom recipe:

http://www.laborjournal.de/rubric/tricks/tricks/trick81.lasso

signal is very strong, sometimes stronger than it should and may improve the sensitivity of your detections.

good luck

i use onlu 10 ug of total protein........we use the novex nupage system and it works well. the detection of antibodies varies. I see signals for some antibodies using milk as a blocking agent and for others BSA. Also for estimating the protein I use the Biorad protein assay which works preety neat. For detection we always use the ecl plus form amersham and its the best in the other options I think. Also if you are using a ccd camera based system for detection then the sensitivity is always less, then you need to move to x ray or infrared( like the Odyssey system).

There are some good suggestions here. It can also depends on your samples. In some treatments, tissues, etc. particular proteins are simply absent, or poorly expressed. Find something that you know will express your protein or use recombinant protein as a positive control. Product information sheets should be helpful.

Also, some proteins are tough to blot. In my experience small, acid proteins are difficult. If possible, test the antibody with immunocytochemistry / immunohistochemistry.

Hello Tiane, I had problems with non fat milk also. Maybe because they increased calcium concentration here in Brazil in non fat milk formulation from 300 mg to 1000 mg. I changed to BSA and it had solved my problem. Good luck!

The above suggestions are good. However, a quick thing to test might be to try a stronger detecting agent such as SuperSignal West DURA from perbio.You could possible ask around; someone should definitely have it. All the best.

Personal experience about this problem. Several possibilities: [1] the primary antibody is not good enough; and [2] low abundant protein can be considered. I have tried a few methods and worked most of the time such as: [1] increase the loading amount of lysate or concentrate the protein concentration; [2] chose another primary antibody; [3] treat the membrane with signal enhancer; [4] increase the concentration of the primary antibody; [5] use more sensitive ECL (such as Femto); and [6] try another blocking reagent (this is more commonly seen in phospho-protein when skimed-milk blocking frequently will not give me signal or gives me very dirty background then try 1% BSA). Good luck.

By the way, I forgot to mention that long exposure is another method I used to do when Femto ECL was not available. I had put my box in 4C for 30 minutes, or even longer, in the old days and it worked a few times. Make sure the amperage you used for transfer is appropriate for the protein. I have used 400 mA to transfer

Article Western Blot: Technique, Theory and Trouble Shooting