Hi Nancy,

so your signal is too strong right? Or is the whole blot dark, like strong background?

The dilution-factor of your antibody normally isn't the reason for too strong signals.

How many µg of protein do you load on gel/well?

Do you make a test before setup a detection time like a 10 sec.-run to see how strong your bands are?

What do you use for blocking? Maybe try 50% Casein in TBST (works good for me, if blocking is a problem) 

Li-cor Westernsure ECL is very sensitive right? So try to dilute it 1:1 with water (Or like 1x solution A(Lu.-enhan), 1x solution B (peroxi) and 1X water), maybe incubate for only one minute (instead of 5), tip one edge of the blot on a peace of paper to drain excess solution thoroughly...

Can you upload a picture of a "dark" blot?

Can you see a brown precipitate on the blot, after incubation with ECL, on the "protein of interest s" expected hight?

The antibody dilutions look okay. Maybe you should properly remove the remaining ECL solution.

I use a waste disposal bag (transparent), cut this at two sides and include the membrane after ECL incubation. Afterwards I remove the remaining solution by stroking over the bag with a tissue paper until all solution is removed (you should add some papers under the bag to soke up the liquid). This results mostly in nice white blots with sharp dark protein bands...

Hello Nancy,

The wash buffer or the washing could be a problem as well. I agree with Simon we need more details of your protocol to help.

1) This could be a problem of blocking the membrane. Check the blocking protocol.

2) Try incubating with a antibody for a protein known to be present in your protein extract. This is to make sure the assay is working.

3) If 2) is ok, try diluting the primary antibody 1:2 and/or decrease the incubation time (or incubate at +4 degree C (cold room or fridge) over night.

Hello Nancy,

I am LI-COR employee and was also involved in the development of WesternSure ECL Substrate. I am rather puzzled by your results.

Were your blots working before? Is this the first time you experienced problem of dark membranes?

The only way your will get all over dark membrane is due to the non-specific binding of the secondary antibody to all over the membrane. This can happen if one forgot to block the membrane or if the secondary antibody is binding to the blocking buffer itself. If the answers to above two scenarios is no, then try adding 0.1 % Tween 20 to the primary and secondary antibody dilution. Also, try diluting your primary and secondary antibody. If you are working with abundant target or loading higher amounts of cell lysates, you can dilute your antibodies further. 

If you provide the details of your protocol, we can help further.

Hi Simon. Yes, the signal is WAY too strong, in less than 5 seconds is very high when I´m developing...I load between 40-60 ug of protein. I didn´t make a test. I block with 5% skin milk in TBST. Here is the picture, it was take at 5 seconds... No precipitate.

Yes, LICOR es very sensitive, I´ll try the dilution.

An update: a coworker give me a SuperSignal West Pico from Pierce, and it did came out rather nice...

Hi Nisha.

Yes, this is the second time I use the LICOR and it was really high signal...

Do you mean it is dark with no specific signal even if you invert the image? We have found that when the transfer pads (used for Wet transfer) are exposed to Coomassie dye, the membrane becomes dark when scanned. For example, if we use the pads to transfer native gels, all blots will become dark ( I mean all subsequent blots). It seems that the Coomassie dye does not get off even if we wash the pads very thoroughly. Also, if you try to label the nitrocellulose membrane using a pen (particularly blue ink), the whole membrane will look dark when you scan it. We avoid writing on the membranes ( say you many membranes and want to number them).

I would load less amount from your lysate (10-15 ug protein) and also try to use a different secondary antibody as you could get non-specific signal.

Good luck

hi

Try blocking with 5% skim milk in TBS rather than TBST ,  dissolve the milk completely whether using in blocking or in antibody buffer and do try increasing your washes with TBST.

Good luck.

Hi Nancy, based on your WB image, it looks like you need to wash the membrane longer. We use 4% skim milk in PBST to wash the membrane, 4 x 10 mins before incubation with ECL. Also, our lab uses WESTERN LIGHTNING PLUS-ECL from Fisher. You may wanna try it. Good luck.

You should increase the number as well as time of washing. We usually do 3 washes of 15 minutes each.

You could improve your blots by increasing the number of washing as said above, for example 4 washes of 10 min each in TBS-Tween 1%, or maybe, you could have a problem of blocking. Do you use milk or BSA for blocking? Usually, 5% BSA is sufficient to block your membrane, in addition to increasing the number of washing.

I wash 3 times with TBST, I´ll try 4 washes. I block with 5% skin milk for 1 hour

After secondary Ab, you can wash 3 times  with TBS, 0.1% Tween, 10-15 min , make a last wash with TBS for remove traces of Tween. I use Pierce ECL Plus  (Thermo)

Good look!

Thanks Liz!!! I`ll try the extra wash, and the ECL from Pierce! Cheers!

Hi

Try using PBS/TBS for blocking with 5% milk and ensure the milk is fully dissolved.

Extra PBST/TBST washes as well.

Good luck!

Rev