Increase antibody concentration.

Try standardizing the western blot with purified protein.

Load maximum tissue sample in the well.

Hi Saswat,

As you mentioned in you question it looks like your transfer was all right. When a particular western does not give signal for target protein there can be different things to trouble shoot. I would look for :

1) Probe for Actin/GAPDH/ or any of the equal loading control to see there is certainly not problem with western / transfer/ antibody incubation/washing steps.

2) if i am able to equal loading control then I can say the western is okay. Now I would check the data sheet that comes with antibody or the webpage for references and find out what kind of tissue or cell people have been used. ( expression of proteins are variable in tissues and cells, so might be your protein of interest is expressed in less amount).

3) for less expressed protein, I use Dura for developing instead of ECL.

4) higher molecular weight protein takes longer time to transfer and vice versa.

Hope you find this helpful.

Good luck

First of all, measurement of protein concentration via Bradford ist useless, when using RIPA for lysis / protein preparation, because you will get no reliable results. Via Bradford you will measure lot of signals from your detergents with almost no influence of proteins on your values...I would propose measurements vial Lowry to get reliable results.

Moreover, in general 20 µg of total protein should be enough, therefore 40 µg should led to quite (too) good results, when doing WB. As also idicated by panceau staining which indicates a well transfer. However, you get no band patterns wen doing development (with ECL and HRP-conjugates Abs?). Furtherore, sometimes you get results and sometimes not. This means you hve to rething different sources for mistakes.

a) the experimentator (do you perform the experiments in each case in the same way?)

b) the technique (is the apparatus working well, are all buffers tested, new and ready to use, is the power suply reliable or is there a possible impact of not well working power supply? etc.)

c) Are all antibodies used tested, working in a reliable manner, new or too old/defect (first and second Abs)?

d) is your detection system working well (age of the ECL solution, concentratiom, have you used all compounds, is there a proble with x-ray fils (age etc.), do you using an other developmental system for visualization and is it woring as expected? etc.))

In my opinion

- you should change the method for protein concentration measurements to get reliable values, then 20 µg should be enough.

- you should try to use an established WB protocoll (e.g. from literature (pubmed) / book (i.e. "the experimentator" series)

- test your components, buffers, Abs to warant wa working method

Try to use positive and negative controls

• Do not dry the membrane after Ponceau staining wash it and keep it wet until blocking step.
• Try to block the membrane with 5% BSA for 1 hour.
• Check antibody dilution according to the manufacturer guidelines for primary and secondary. As you mentioned that some time you find the band and some time not that is comes with the antibody standardization issue.
• Primary antibody incubation should be overnight at 4 degree.
• Check the pH of the washing buffer (TBS-T or PBS-T) and also insure that Tween-20 concentration is not more than 0.05%.
• Wash 3-4 times, each washing should not more than 5 minutes.
• Avoid over-washing after antibody incubation.
• Prepare ECL just before the development of the blot.

Best of luck..

I have several suggestions for you.

(i) If you are not able to see bands with a reversible membrane stain like Ponceau means there is no transfer of proteins on membrane and/or that stain needs to be made fresh.

(ii) You may want to try much more sensitive, dependable and western friendly reversible membrane stains like "BLOT-FastStain" (manufacturer: G-BioSciences).

(iii) You may also want to look into our membrane protein stain as an alternative protein assay. ( Heda G.D., Kunwar U, Heda RP. A modified protein assay from micrograpm to low nanogram levels in dilute samples. Anal Biochem, 445C, 67-72, 2014).

Hoping these suggestions may help. Good-luck.

Rahul Vishvkarma Thank you for your response. But every where it was mentioned that Tween 20 should be 0.1%. So is it fine to take 0.05%

Tween20 is a mild detergent. Its' concentration depends on the sensitivity of protein of your interest. You can try both and see which one works out the best for your protein.

Hi, Saswat KUMAR Mohanty.

Have you noticed If you're always getting bands just after sample preparation and maybe when you use the same sample again, it vanishes? If so, maybe, your proteases and phosphatases are not working after homogenization and you're having proteolytic activity and then loosing your sample.

Just another possibility.

If possible, let us know what happened when you solve your problem.

All the best.

Saswat KUMAR Mohanty Yes 0.1% is recommended but if your bands not coming after taking all necessary precautions it may be issue of the higher detergent concentration and duration of washing. Once I have also face the same problem.