12 replies to this topic

[Leon_K]

Hi, i've just performed my first western blot with the help of a colleague at the lab. Unfortunately, things didn't go as smoothly as expected. I've ran into a few problems while performing my WB.

1. My colleague forgot to add SDS while preparing the gels. How would this affect my bands and their separation?

2. While incubating with my anti-mouse secondary antibody in an orbital shaker, a lab member turned up the temperature to 37 degrees celsius. The manufacturers instructions for incubation temperatures is at room temperature.

With all these in mind, my samples showed a bit of diffuse signal across the entire lane with a darker, more concentrated band somewhere in the middle. This band isn't very well defined either with fuzzy edges. My negative controls however, did not show anything on the lanes.

Also, would the lack of SDS in the gels affect my marker migration and separation? The bands seem a bit 'off'.

Your advise/input is very much appreciated. Cheers  

[Prep!]

Hi there... ya withough SDS in ur gel the technique is no longer SDS_PAGE... its more like a native gel with a SDS_PAGE sample buffer... tat is the precise reason for all the wierd observations.. just repeat the exp with proper gel preparation and i guess u will be fine with it!!!
Now room temperature is a very controversial term.. here in india RT can be 37 degrees!!  

Best luck!!!

[mdfenko]

sds in the running (electrode) buffer will migrate through the gel so it is still sds-page (this is how the pharmacia phast gel system worked). the sds front will pass the proteins as they stack. in a small gel (like the phast gels) the defects may be negligible but may not be in larger gels (this is pure speculation on my part, i've never tried it).

but, yes, try again with a properly prepared gel.

[Prep!]

i agree!!  

so the best bet is running the gel again with properly prepared gel and samples!!!  

[Leon_K]

Alrighty, i'll try that this week and get back to you guys! Thanks for the advise.  

[Leon_K]

Okay, i just ran a second experiment, essentially the same as the one mentioned previously but with SDS added into my gels. I still have the same problem of background staining all along the lanes. I think there might be a problem with unspecific antibody binding.

The primary antibody used was a mouse anti-human polyclonal. Made up in 2.5% BSA and incubated at 4 celsius overnight.

The secondary antibody was an anti-mouse HRP conjugated, raised in sheep. Incubated for 2 hours at room temperature (~22 celcius) and made up in 2% milk/TBST.

I've attached my result if any of you would care to have a look at. This was imaged with iQ350 imager with a 15 minute exposure using ECL. I should also note that the signal emitted was rather low.

What could i have done wrong? Helps, suggestions are most welcome  

Attached Thumbnails

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[mdfenko]

why do you change blocking agent between antibodies? with what do you block the membrane?

you may want to consider adding normal sheep serum to the block and antibody solutions.

[Prep!]

mdfenko, on Jul 1 2010, 07:57 PM, said:

why do you change blocking agent between antibodies? with what do you block the membrane?

you may want to consider adding normal sheep serum to the block and antibody solutions.

yeah i agree.. sorry if i missed it but i dont see any blocking step!!!
ideally its better to clok the blot before the addition of primary antibody!!! and u can use the same solution to prepare the primary and seconday!!! why switch from a BSA to a 2% milk!!!

[Leon_K]

I blocked it with 2% milk in TBST.

Good point, my boss asked me to make up my primary in BSA which is odd since i am blocking with milk. I am planning to re do the experiment next week.

Btw, i am quite unfamiliar with WB, how would the use of BSA or milk affect my results? Some people in my department use milk, some use BSA.

Thank you!

[mdfenko]

Leon_K, on Jul 2 2010, 05:05 AM, said:

how would the use of BSA or milk affect my results? Some people in my department use milk, some use BSA.

you use different blocking agents for different purposes (although most blocking agents are pretty general in use).

for instance, i wouldn't use milk to block if i was probing for phosphoproteins (some do but their antibody may be more specific). milk contains casein which is highly phosphorylated.

i also wouldn't use milk with a biotinylated system. it also contains biotin.

bsa is a general use protein block which can be used where milk can't (and where it can).

we also add normal serum from the species in which the secondary antibody is produced.

[Gerard]

I've never seen this kind of crackling as in the picture thus i'm guessing.
I,m not sure but looking at the picture i have the feeling that the problem originates from the gel casting or the imager. I don't think the blotting or the probing caused this problem.

[Leon_K]

mdfenko, on Jul 1 2010, 06:27 AM, said:

why do you change blocking agent between antibodies? with what do you block the membrane?

you may want to consider adding normal sheep serum to the block and antibody solutions.

I use BSA with my primary Ab because we re-use our primary antibodies after probing. Sodium Azide is added to it to preserve it for future use.

[Leon_K]

Gerard, on Jul 2 2010, 09:59 AM, said:

I've never seen this kind of crackling as in the picture thus i'm guessing.
I,m not sure but looking at the picture i have the feeling that the problem originates from the gel casting or the imager. I don't think the blotting or the probing caused this problem.

You're right, after help from a colleague, it seems that i've used the wrong settings to image the blot. Also, i used regular ECL which gave a very weak signal. After using Super ECL, the bands were much clearer.