Use 5x or 6x dye for loading. Use 1.5mm gel with 10 well comb. Boil the sample for 5 min with eppendorf lids open (lid open for all samples: the samples should be made to equal volume by adding lysis buffer before boiling to justify equal evoporation). Of note, 30ug is sufficient for good Westerns.

Hi Dalia,

Are you actually asking about working out the concentrations? Let’s consider your current sample 1 is of about 40ug/40ul (or 1ug/ul). As you will add 1/5th or 1/6th of the final sample volume the DNA loading dye (depending on the type of dye you have) and the rest will be your sample, you will end up with 32ug/40ul or 16ug/20ul. This means you may need to concentrate your sample roughly 2-fold before proceeding.

The sample 2 may therefore need to be about 4-fold concentrated. Is your last sample really in ug/mL?.. therefore 1000x less concentrated than the sample 2?

If you have trichloroacetic acid (TCA) in your lab ... Transfer a volume equivalent to, for example 100 ug of protein for each sample to a clean microfuge tube. Adjust each to 10% TCA using a 100% TCA stock. Leave on ice for 30-60 min. Centrifuge at max speed in a microcentrifuge at 4 deg C for 15 - 30 min. Carefully aspirate of the supernatant. Do not disturb the pellet. Resuspend at 2 mg/ml in 1X Laemmli sample buffer. Buffer will turn yellow (pellet contains acid). Add 1M Tris base solution, 1 ul at a time, with gentle mixing, until solution turns blue. Mix. If turns yellow again, add a bit more tris base. Heat to 70 deg C for 90 seconds. Mix. Centrifuge to pull evaporated/condensed water back down into base of tube. Mix. Load required amount onto gel. Load same volume of 1X buffer into empty wells (if any). Be careful when handling the TCA!!

thank u

I usually use 10 to 20 ug of protein.

use 10well gel, 1.5mm thick.