Lysis and Sample Preparation

The beginning of any western blot starts with cells or tissues. First, the protein from these cells must be solublized with a strong detergent. The best and most common buffer to solublize protein for western blotting is Laemmli sample buffer (Named for Professor Ulrich Laemmli).

Laemmli Loading buffer:

• 60 mM Tris-HCl pH 6.8
• 2% SDS
• 10% Glycerol
• 5% β-Mercaptoethanol
• .02% Bromophenol Blue

To make 10 ml of 3x Stock of Laemlli Loading Buffer

• 1.8 ml of 1M Tris-HCl pH 6.8
• 3 ml of 20% SDS
• 3 ml Glycerol
• 1.5 ml β-Mercaptoethanol
• 700 ultrapure H20
• .006 g Bromophenol Blue

Required Materials and Reagents:

• Laemmli loading buffer
• Protein source (cells or tissue)
• Bunsen burner
• Steel water bath
• Eppendorf Tubes
• Tube rack for boiling
• Table-top centrifuge

Protocol:

1. Add Laemmli buffer to your samples so that the final protein concentration is approximately 1 ug / ul. The protein mass will vary between cell lines and tissues, so it is best if this concentration is determined empirically by a total protein assay (e.g. Bradford Assay, BCA assay).
2. Use the tip of your pipette to mix the Laemmli buffer around the well and scrape the bottom of the well to solublize all cells and proteins.
3. Draw up the Laemmli buffer with your pipette and place into a clean, autoclaved eppendorf tube.
4. Boil the protein samples for 5 minutes in a water bath. Make sure that there is something to put downward pressure on the lids of the eppendorf tubes. If not, the heat will increase the pressure inside the tubes to such an extent that the lids will pop off and water will get inside.
5. After boiling, spin the samples in a table-top centrifuge at approximately 15,000xg for 3 minutes.
6. Let the samples cool to room temperature before loading the gel.

View our protocol for casting SDS-PAGE gels.