Dear Sokviseth,

I found answers for a similar question, you may be interested:

https://www.researchgate.net/post/Why_am_I_getting_diffused_bands_in_western_blot10

You may also want to have another look at the troubleshooting on some websites such as rndsystems and bio-rad:

https://www.rndsystems.com/resources/technical/troubleshooting-guide-western-blot

https://www.abcam.com/protocols/western-blot-troubleshooting-tips

https://www.bio-rad.com/en-cn/applications-technologies/troubleshooting-western-blots-with-western-blot-doctor?ID=MIW4HR15

https://precisionbiosystems.com/western-blot-troubleshooting-guide/

Sincerely,

Dear Sokviseth,

This troubleshooting guide might help you:

https://www.abcam.com/protocols/western-blot-troubleshooting-tips#high-background

From reading your questions, you probably meant to say that you loaded 20 ng of protein. You didn't write about a blocking step after the transfer or washing steps after antibody incubation. This could already be a possibility of causing this high background/ diffuse bands.

Best wishes

Hi, did you stain with ponceau? Did you see if the band are diffuse, blurry or have some problem in general? Did another coworker use the same antibody? What type of tissue did you analise?

As I can see your GAPDH is ok bu your LC3 signal is not only diffuse but aspecific so you have to understand if is a samples, running/transfer problems or if the Santa Cruz antibody work well or not.

You can try to use a positive control for LC3 different from your samples, as cell culture, run a gel to understand if you have a clear signal for your protein

Do you prepare your own geles? Are fresh solutions or from old stock? What time these polymerize? What % of polyacrilamide are? What size? How potency you use for the running? All these are few questions to address and to understand the issue may happen to get diffused bands. Can you answer?

Youssef Siblini Thank you so much! Yeah those information is helping me a lot.

Janine Lux Thank you so much! After blocking, I added primary antibody (in 1% skim milk) to the membrane and incubated at 4 degree celcius for 48H. After 48H incubation, I was membrane 5 times with 10 minutes each and then added 2nd antibody (in 1% skim milk) and incubated at room temperature for 1H. After that, I washed membrane 5 times again with 10 minutes each.

Valeria De Arcangelis Hi... I didnt stain with ponceau or any things.

And someone said that my GAPDH bands are too thick, do you have any ideas how can I fix this?

Xochitl Ambriz Peña For gel, we bought it from SMOBIO (Q-PAGE Precast gel). All solutions and buffer are freshly made just one day before the experiment.

Your GAPDH bands look ok, perhaps you could sharpen them a little by using a gradient gel or by using a greater dilution of antibody, but that is not really required.

Your LC3B staining is a different matter. Either

• your antibody is very unspecific, staining many proteins
• your protein is degraded and you are detecting proteolytic fragments (what is the molecular mass of your protein, all fragments should be smaller)
• Your conditions for AB staining are wrong, so that you get total protein staining. Have you blocked the membrane before staining? Are you dealing with any detergents other than SDS somewhere?

Hi, first of all it would be a good choiche to stain with ponceau your next membrane ( and with coomassie your gel after the transfer; it is a simple procedure and this can help you to control the different step of your experiment).

You have to understand if you have a problem during the run, the samples lysate (can I ask if cell culture or animal tissue???) or the transfer.

Honestly I am sure that with precast gel you dind't have run problem but, for me, 2h hour of thansfer (can I ask tank or semi-dry?) for a 15KDa problem it is too long. I generally use a tank transfer and a constant voltage all on ice, so I dont' want to give you wrong advice on time and Amp, but you can decrease the transfer time. Maybe you have problem only with small proteins as LC3 during transfer, maybe too hot, maybe too long; you have to try loading only few samples or a positive control for LC3.

Why you incubate for 48h (is it correct or a typo?), you choice to dilute a lot your antibody (1:4000 respect the datasheet recommendation) and then incubate so long. Use a standard overnight signal 1:1000, and as Dr Engelbert Buxbaum said you can improve the wash step.

If you continue to have same problems and your lab have money think about find from letterature another LC3 antibody. Your antibody is from Santacruz and generally their antibodies can be really good or a totally disaster!!!!

You didn't write the incubation dilution for GAPDH but if you want to modify the thickness you have to dilute the antibodies, and I will try with the secondary. I think the signal is good but if you want to improve the normalization try it, after you resolve your principal problem with LC3.

Good luck

Hi Sokviseth,

I agree with Engelbert Buxbaum .

My main suspicion is that you just got a bad antibody. We are really into WB, and some companies really sell many antibodies that don't work all too well (to put it mildly). So, what you need is a positive control. Some condition someone else has shown before to induce LC3, or cells that express it at high levels. (AB suppliers will use these positive controls). There is a possibility that, at least under your experimental conditions there's no LC3 expression in the cells, right?

Since your GAPDH looks good, you could try a couple of other established stains (if possible at similar weight, to rule out the too long transfer possibility) to sort of prove your LC3 AB isn't the best.

Good luck!

change antibody

Antibodies should be validated (see doi:10.2144/000113382), ask your supplier whether that has been done with the AB you are interested in.