3 ideas come to mind:

1. You could try blotting with more protein. I usually use 20-40ug/well, using a 10-well comb with teeth that are about 5mm wide and 2mm deep. (Don't use combs with teeth that are too small or you will overload the lanes.) I find that for some of my weaker antibodies, it's necessary to use 30-40ug of protein to detect any signal at all.

2. You could try blocking with a different blocking solution. Some antibodies work better in 5% BSA than in 5% NFDM (although the manufacturer will usually indicate whether this is the case).

3. You could try blocking 1 hour at room temperature instead of 4 hours. According to Cell Signaling Technology, "Blocking the membrane for too long can obscure antigenic epitopes and prevent the antibody from binding."   http://www.cellsignal.com/common/content/content.jsp?id=western-trouble

Also keep in mind that some antibodies will not work well for your system, no matter how much time and effort you spend trouble-shooting them. In our lab, we usually purchase new antibodies in the "sample size" amount and give them 3 or 4 good tries to work, varying the antibody concentration and blocking buffers. If they don't work after all that, we can often call the company and have it returned for free and then try another one.

Best of luck with your blotting! I'd be interested in hearing how it turns out.

Hi Jillian, thanks a lot for your kindly help..I also think about some changes that I will try next time.

1. I want to load more protein but it's hard to obtain large amount of protein from CD8+ naive/ memory T cells or tumor CD8+ T cells :(. However I will try with 20ug.

2. Switch/ reduce % of blocking reagent: Now I'm using TBST 5% skim milk for blocking 4hours, Ab is dillute in TBST 1% skim milk. As your recommendation, I will block in TBST 5% BSA or  TBST 1% skim milk for 1hour, RT.

3. Increase primary and secondary Ab concentration.

I will inform you how the new result turns out ^^. 

Sincerely,

Hi Hanh Hong,

I've never tried diluting the blocking buffer before. I always use either BSA or NFDM at 5% in either TBS-T or PBS-T, so I'm not sure if reducing the percentage will help.

Also, I would be careful about increasing the concentration of the secondary antibody too much, because it may just cause a lot of non-specific binding, without increasing the signal for your primary antibody. If 1:5,000 works for the other primary antibodies you use in your lab, then you may want to stick with that.

I'm looking forward to hearing how it goes!

Best,

Jillian

Hello Jillian, 

I'm so sorry that I have to postpone all experiments due to our coming Lunar New Year. I will restart Western next week. Could you please give me your email/ facebook... so we can contact later? 

1. In Abcam protocol book, 'Trouble shooting for Western blot', they recommend that too much blocking does not allow us to visualize our protein of interest. " Instead of using 5% milk in the antibody buffers, try removing the milk or using 0.5%. Switch blocking reagents or block for less time".

If Primary antibody has the phosphate tail, we should use BSA instead of skim milk.

2. Do you know why do different companies (even same company) offer different sizes of one protein? I attach 4 products from Abcam and Santa Cruz, in which predicted molecular weight of Foxp1 can be 38kDa, 75kDa or 85kDa. It really makes me confused.

Sincerely,

Hanh.

The band size variation between 75-85 kDa is possible due to different MW markers and running conditions. The abcam antibody showing the 30-40kDa band is a western against the recombinant peptide which they used as immunogen (not cell-lysate). So you should expect your band around 75-80kDa range. Good Luck.