7 replies to this topic

[cm13]

Help me please with my Western Blotting!
Here's my protocol:

Quote

Acrylamide Gel: 10%

4mls water
2.5mls 1.5m tris ph 8.8
3.33mls acrlyamide bis 29:1 ratio
100ul 10% SDS
50ul 10%APS made up on the day.
5ul TEMED

Topped off with layer of Isopropanol.
Waited 40 minutes for gel to definitely set.
Poured off the isopropanol, and gently washed out with water.

Stacking gel:
3.05mls water
1.25mls 1mM Tris ph 6.8
650ul of Acrylamide bis 29:1 ratio
50ul 10% SDS
50ul 10%APS
5ul TEMED
Waited again for 40 minutes for gel to set.

Sample loaded consisted of:
11ul of Cell lysate sample
4ul of Sample buffer (taken from 1ml of 5x Buffer with 125ul of mercaptoethanol added)
5ul of distilled water.

(only 1 sample was loaded, and a ladder was loaded too)

The sample was run at 80volts for 20 minutes before being increased to 100volts until the sample buffer appeared to be at the bottom of the gel and the ladded was visible.

It was at this point I thought there could be a problem, as the sample seemed to diffuse a little out from the well and appear like a large spot stretching out over 2 wells.

Nonetheless, I still continued with transferring (for 2 hours at 80V).
Afterwards, a ponceau stain showed there was something present.

I blocked the nitrocellulose in 5% milk powder in TBS-Tween for 1 hour.
Then I washed in TBS-T for about 5 minutes.

The primary antibody ( 1:1000 dilution of a Myosin Light Chain 2 antibody in 5%BSA in TBS-Tween) was added and the sample was left overnight shaking at 4C.



Next day, the membrane was washed 3 times for 5 minutes each time in TBS-Tween.

The Secondary antibody (an anti-rabbit antibody diluted 1:1000 in 5% BSA in TBS-Tween) was then added and the sample was left shaking at room temperature for 1 hour.

The membrane was washed again 3 times for 5-10 minutes each time in TBS-Tween.

The membrane was then developed in Pico stain and being placed in s-ray film for approximately 5-10 minutes.

Yet, nothing appeared on the x-ray film.
This protocol has worked in the past for other people in the lab, and all the developing stuff got results on the same day for other people so it's not faulty equipment that's hindering me.

Does anyone have any suggestions of where i could be going wrong? I would really appreciate some help, as i've tried altering the protocol in different ways yet still have no result. It's getting really frustrating....

[little mouse]

every thing looks fine until antibody incubation.
During migration it is normal that you see a line for the front migration, going from the first to the last sample, and not single individual front line right under the well. It's nomal that you have a latteral diffusion of bromophenol blue. You still have restricted bands of proteins.
You saw by Ponceau red staining that proteins were transfered. This is fine. (sometime you don't see it, but it's still OK as antibody staining is much more sensitive than Ponceau red staining).
I'm just surprised by the blocking steps.
You first incubate your membrane in 5% milk (I hope it is non fat milk), and then you incubate your antibody with 5%BSA ? why do you change?
by the way 5% BSA is quite a lot. Usually I use 1%

My protocol is :
block 30 minutes to 1 hour in TBS (without Tween, but you could add it) with 3-5 % milk OR 1% BSA, depending on the antibody.
then incubate with antibody in TBS with 0.3-0.5% milk or 0.1% BSA.
wash with TBS plus 0.05% tween-20 three times after each incubation with antibodies.

[AVEA]

After looking at the protocol the main thing that I see is that you are using a 1:1000 dilution of the secondary. Typically the secondary antibody stock is ~1 mg/ml this means means that you are using ~1 ug/ml. This states that you are using too much HRP with Pico. Typically Pico needs a concentration of 10-50 ng/ml. I usually use 10 ng/ml secondary. I know this sound strange since you are not getting a signal however if you use too much HRP then the signal will deteriate before you are able to develop the film. Go to the webpage http://www.piercenet.com/files/TR0024-Opti...b-dilutions.pdf to obtain more information on optimization of the antibody dilutions. Also in the isntructions for SuperSignal WEST Pico it states the range that needs to be used to obtain a good signal. I would highly recommend that you decrease the amount of secondary antibody (10-50 ng/ml) and you should get a signal. If you secondary concentration is 1 mg/ml this means to use a 1:20,000 - 1:100,000 dilution. I typically use a 1:100,000 dilution.

[cm13]

little mouse, on Aug 6 2009, 01:50 PM, said:

You first incubate your membrane in 5% milk (I hope it is non fat milk), and then you incubate your antibody with 5%BSA ? why do you change?
by the way 5% BSA is quite a lot. Usually I use 1%

My protocol is :
block 30 minutes to 1 hour in TBS (without Tween, but you could add it) with 3-5 % milk OR 1% BSA, depending on the antibody.
then incubate with antibody in TBS with 0.3-0.5% milk or 0.1% BSA.
wash with TBS plus 0.05% tween-20 three times after each incubation with antibodies.

It was non-fat milk. That's what the protocol i was given said. I had tried also with BSA as a blocking solution before, but that didnt work out either. Does the blocking solution have to be the same as what the primary antibody is diluted in?
And what difference would 5 or 1% make to the western?
I'm only a beginner at all of this.

[little mouse]

by using too mych blocker you might have troubles if your antibody is weak. But AVEA pointed something important, I didn't see it, you use too much secondary antibody. I also use 1:10,000 to 1:100,000 (from 1 mg/mL stock)

[cm13]

I just checked there about my secondary antibody. The stock i took it from was a 500ug/ml solution. Is that still too much?

[AVEA]

cm13, on Aug 6 2009, 08:44 AM, said:

I just checked there about my secondary antibody. The stock i took it from was a 500ug/ml solution. Is that still too much?

That seems like it is to much. You are using 0.5 ug/ml. I would suggest that you use a 1:25,000 to 1:50,000 dilution of your 500 ug/ml stock solution and you should not have any problems.

[madrius1]

Any chances your primary antibody has gone old?

You could try with a working ab, such as actin, gapdh, tubulin, or anything that is still working and that you know will be present in your sample.

Is it possible that your cells did not express any Myosin Light Chain 2 ?

I also agree 1:1000 secondary ab is too much.