3 replies to this topic

[lipidlover]

Hello all,

I have two questions, first:

I have recently been developing a protocol for western blotting all tyrosine phosphorylated proteins from B cell whole cell lysates. I have optimized the antibody concentrations and incubation times and now I am having a problem of inconstancy. Basically the band intensity seems to depend on where it is on the blot.

Using cleared whole cell lysates, frozen stocks.
Determined total protein concentration with BCA assay, serial dilution of BSA standard in identical lysis buffer
Loaded equivalent total protein on gel in Laemelli, determined by results of BCA assay
Loaded identical samples in duplicate, on opposite sides of gel
Transferred in OWL system, 260 mA for 1 hour, confirmed minimal blowthrough using second membrane
Blocked overnight in 4% BSA at 4 deg C, stained with primary for 3 hours at RT, and secondary 1 hour at RT, using PICO ECL.

All the bands on one side clearly had a higher intensity than the identically loaded bands on the other side.

Some things I am considering:
1) Transfer apparatus is transferring unevenly
2) antibody incubation needs more buffer to evenly cover blot (I don't think this is it because the blot is essentially floating and freely moving during agitation
3) the ECL substrate is not agitated while incubated. Could this be the issue? Are the blots commonly agitated while incubating with ECL reagent?

I am hoping to either reprobe the blots with different antibodies, or use a Ponceau stain to see if the protein amounts are equal. If anyone has any other ideas on how to determine the cause and or directly fix the problem, I would love to hear. Thanks.


secondly:

I want to look at the B cells at precise timepoints after stimulation. This is impossible using my current method of spinning down stimulated cells and resuspending in 1x RIPA buffer with phospho and protease inhibitors. Do people use concentrated lysis buffers to directly lyse suspension cells in media/other buffer instead of spinning into lysis buffer? Any input is greatly appreciated!

[bob1]

You are definitely on the right track with the ideas you have.  These problems are usually in the transfer or later stages.

Ponceau or coomassie stain of the membrane is the best option for looking at the transfer - if it turns out to be this, it may well be that the tank has a small leak, replacing the gasket may solve this, but you can try keeping it closed with rubber bands temporarily.

I don't normally agitate in ECL, but there is no reason why not.  Just make sure you use enough volume to totally cover the membrane, 5ml per 60 cm2 membrane works well.  You could also incubate in a sealable bag.

[GNANA]

As bob1 said ponceau staining would clarify whether the problem is before or after probing...just thot to add another possibility, if your ponceau staining shows you even transfer then have a check in your probing cassette...sometimes if the cassette doesnt close or press the filter evenly throughout the intensity of the signal may vary ..so try in another cassete or in the same cassete at another place or direction ....

[EvilTwin]

How do you incubate the membrane with the Ab? If ponceau staining shows that the problem emerges after the transfer, there are still 2 possibilities I can think of. GNANA is right - make sure that the casette closes tight. The other cause may be an uneven incubation with antibody. Do you use a roller or a rocker? Make sure that your container is placed properly (meaning that even if the device doesn't move evenly, you will  get different intensity from top to bottom, not from left to right) or try reversing the membrane/container by 180 degrees, say every 30 minutes. Well, it helped me solve 1 of my 999 problems with WB, not that I am a WB expert :-) ,  but it might help and requires nothing except your attention.