FAQ
What blocking agents can be used with ECL Western Blotting Detection Reagents?
We recommend the ECL Blocking Agent at 5% dilution in PBST or TBST (0.1% Tween 20) buffer.
We have also found several other blocking agents which work well for ECL Western Blotting on Hybond ECL and Hybond-P membranes. All produced similar sensitivities with only slightly variable signal-to-noise ratios. These chemicals (in PBST) are listed below in descending order of efficacy.
1. 5% Liquid Blocking Reagent for 30 minutes; equivalent or superior results to 5% milk.
2. 3% Bovine Serum Albumin (BSA) for 30 minutes.*
3. 1% Liquid Blocking Reagent + 0.5% BSA for 60 minutes.
4. 2% Fish Gelatin for 60 minutes.
5. 5% Normal Goat Serum for 60 minutes.
Note: BSA is the optimal choice when working with phosphorylated proteins.
Tips for choosing a methanol concentration for protein transfer buffer
Methanol that is included in the Towbin transfer buffer is necessary to achieve efficient binding to nitrocellulose and PVDF (Polyvinylidene difluoride) membranes. Methanol is not required for binding to nylon membranes. Methanol improves protein binding by removing protein-bound SDS (Sodium dodecyl sulfate). However, methanol may cause a gel to shrink resulting in a decreased protein elution rate from the gel. Therefore, the recommended methanol concentrations are:
Membrane type | Methanol % |
Charged Nylon
|
0
|
Nitrocellulose | 20 |
PVDF | 15 |
I often have spotty membranes. What can be causing that?
It can be that the blocking agent is not completely dissolved, resulting of protein "lumps" on the membrane that antibodies or reagents bind or interact with. Or, if small pieces of gel pieces still remain on membrane this can also result in spots.
I handel a lot of blots in my lab every week. The washing procedures take long time and I have a feeling I do not treat my membranes so similar. Sometimes it is 15 minutes wash and sometimes it is 25 minutes. Will this affect my results?
It is time consuming with blocking, probing and washing of membranes. It is hard to treat the identically, which may affect results, such as background and signal intensity. We actually have an instrument, Processor Plus, for automated processing of membranes that will handle 4 blots simultaneously and suitable times can be set for the different steps.
Why do I get a negative image on my Western blots? The bands are either white, clear or outlined in black with a low to moderate gray background across the blot.
Excess reporter enzyme (horseradish peroxidase) in a small area can rapidly exhaust its substrate in the short time it takes to move the membrane from the detection reagents to film (or a CCD camera or scanner).
You may be able to visualize your bands by returning the membrane to the detection reagent solutions for 5 – 10 minutes and then repeating the detection. The long-term solution is to reduce the secondary antibody dilution (or whichever antibody is conjugated to the reporter enzyme) or to load less protein onto the gel, thereby decreasing the amount of target.
How do I know that the membrane is low fluorescent? And, can I use the same membrane for 2D western?
How do I know that the membrane is low fluorescent? And, can I use the same membrane for 2D Western Blotting?
We have 2 low fluorescent membranes that we recommend for ECL Plex. One NC, Hybond ECL and one PVDF alternative called Hybond LFP. You can try other membranes, but then you have to test them first. If they give elevated background, there is risk that you loose the weak signals. And yes, you can also use them for 2D Western Blotting.
The pore sizes of membranes differ, when should I use membranes of smaller pore size?
The standard pore size is around 0.45 µm and works well in most situations. If the targets protein is small, down to 5kDa, we recommend using membranes of 0.2 µM pore size.
How can I check whether my ECL reagents are working properly?
You can perform the "blue-light test." Simply combine equal volumes of Detection Reagents 1 and 2 in a microcentrifuge tube. Mix, and add several µL of an HRP-conjugated antibody that is known to be functional to the tube. Mix well and turn out the lights in a dark room. Functional reagents will begin to glow bluish-white fairly rapidly.