air bubble while loading sample.

[IoDiNE]

Hi,I am new here.I would like some help with WB.I am using b-actin as internal control and load all samples in the lane as gently as possible.But my b-actin bands are not equal in all samples.I have measured the protein concentration also and load equal amount of samples in the lanes.My boss feels my pippetting may not be perfect,though at times an air bubble comes out of the pippete tip when trying to push the last remainng sample into the lane and pushes the sample a little above the lane.what is the best way to load the samples in the lane or good pippetting techniques.

[admin]

Hello there!

Yes I hate eppendorf pipettes because of this reason. If you have the blue ones (I forget the make) they eject VERY SLOWLY and so you dont get any puffing or air bubbles in your samples.

Also make sure you check your loading buffer. Usually sometimes there is not enough sucrose to keep the samples down. I make a special buffer with more sucrose to keep my samples falling like a rock to the bottom. This is helpful when you are comparing samples.

To fix this even though you are loosing samples, simply divide the OD (optical density from a densitometer):
Your protein band in Lane 1 (band values) divided by your internal control OD (eg. b-actin) value in Lane 1.

This way you can compare to:
protein band in Lane 2 (band values) divided by your internal control band (eg. b-actin) OD value in Lane 2.

However if submitting to a journal it is much better to have at least 1 gel that is perfect in loading of beta-actin (internal control and loading control).

Cheers!

[Doc]

Hello

Yes air bubbles are irritating. However just make sure that you load slowly all the way. I load until the first stop on the pipette, then slowly and carefully withdraw the load tip slightly up (but keep in inside the well!!), push gently the rest of the sample out. If you do it slowly the bubbles will now end on top of your sample and will eventually leave the well. The best is to do it so the bubbles comes out one at a time. The time you use here, will save you for a lot of work later.
To check out you pippeting technique, try to stain a coomassie first.