Why don't you try to use RIPA buffer for lysis? Transferring can be done at 350 mA for 100 min. For conjugation with primary and secondary Abs you don't need 5% milk. only 10 ml of 1xTBSt for 1 uL of Ab. Skim milk is only used for blocking step. Hope you will find your mistake.

When I performed semi-dry transfer, I recalled the transfer time was less than 20 mins. It seems to me that your conditions for transfer are too extreme.  What machine do you use for semi-dry?

Hi Neda,

I have to agree with Mirian on the transfer time. We routinely perform western blotting for proteins between 10 to 250 KDa using Bio Rad turbo blot for 7-10 min at 18-20V on a PVDF membrane. Also, your blot shows a lot of non specific signals around the edges - is it because you have uploaded maximum exposure time or you still have the nonspecific signals at lower exposure time? If you don't mind please let me know the answers for the following questions:

1. Which loading dye did you use?

2. Why do you store your protein samples at -20C? It should be stored at -80C.

3. Why are you not using prestained protein ladders? It will help you resolve a lot of issues, for instance if the transfer is complete!

Let's start with the gel. Stain the gel with Coomassie after transfer. Do you see any protein on it? For semi-dry transfer, ensure there are no bubbles and that all parts of the transfer sandwich are wet. Remember to avoid methanol if the membrane is nitrocellulose.  After transfer: Use a Ponceau or Pierce stain before you apply your primary antibody. Do you see protein? 50ug is a lot of protein, so you should be able to see bands. Make sure you have successfully transferred protein from the gel to the membrane. Regarding the antibody: What protein does the antibody detect? Do you have a positive control? For example, if you are studying Hsp70, one of the lanes on your gel should contain a whopping amount of purified Hsp70. This way, if you get no signal, you will know it is because of an antibody problem and not because the protein is simply too low in abundance.

I would suggest to stain the gel with Coomassie, if you haven't.

Good separation on the gel is the very first step of get the western working.

I'm writing this because;

Ponceau stain the membrane after transfer, but before you block it. To remove the Ponceau stain you can use HBSS while rotating for 5-10 minutes (however long it takes to get rid of the stain on the membrane).  The Ponceau will show how your transfer of proteins went onto the membrane- if the transfer was successful.  The Coomassie staining of the gel just shows if there are any proteins still left on your gel after transfer.

Is there methanol in your transfer buffer? I always do 60 mins at 100V. It has to be done in COLD conditions so the buffer and all liquids should have been kept at 4 degrees, or the water made to use the buffer kept at 4 degrees.

Dear Mirian Mendoza, We use the Bio-rad Trans-blot Sd Semi-dry Transfer Cell 170-3940 25v 2a,

Dear Damian,

After transfer if gel still good to work with it. To do comassi? It will be a goo idea ,If I see my protein on gel, it means no transfer happen

I agree with Mirian, we use the same semi dry system an we don't go more than 15-20 minutes at 18-20 V for the transfer. You should Ponceau stain the membrane before blocking. Also i did not notice a boiling step in your protocol. Without adequate breaking that the protein may still not have crossed the stacking gel. What loading buffer did you use? Did you have a  boiling step?

Did you heat the protein sample before loading ?