Gel Loading Calculator for Western Blotting

This is something I made which has been a huge help when running western blots. It will take samples at any concentration and normalize them all to each other, making prep for loading your gel a breeze.

The beauty of this is you can easily mess with any parameter you'd like without having to redo any math.

Just edit the red parts and the green shaded columns are your aliquot volumes.

File Link: https://docs.google.com/spreadsheets/d/1DxwFme3zP4Jw8HLSPWuod7Rc72npsZvv/edit?usp=sharing&ouid=111441827851825405038&rtpof=true&sd=true

Access to the file for editing is locked but if you download the file you can edit it on your own computer.

Here's the calculator:

I'll take you through how to use it, step by step:

Step 1: Run BCA or other protein quantitation assay on your lysates

A protocol for running the BCA itself can be found here: https://www.researchhub.com/post/2000/bca-protein-quantitation-assay
Any protein quantitation assay can be used, I personally prefer BCA

Step 2: Input Your Values

Columns 1 and 2: Fill with sample descriptions of your choosing, such as cell line, treatment, etc. Whatever you'd like to put in to identify each sample, you have 2 columns to do it.
Column 3: Input your protein concentration values for each sample here in ug/uL.

Step 3: Input Values into Calculator Bottom Section

These values are specific to the type of gel you will be running, I will go through these one by one:

Reducing Agent Dilution Factor
- I use 1M DTT as my reducing reagent, with 50mM final concentration in my gel-ready samples. This is a 20x dilution, therefore I filled in a 20 here. This may be different depending on what you're using. If it's 10x reducing agent, then put a 10. etc.
Laemmle Buffer Dilution Factor
- My lab uses 4x Laemmle buffer stock diluted to 1x final concentration in my gel-ready samples. Therefore, this is a 4x dilution, so I filled in a 4 here. As above, this may differ depending what your lab uses. If you're using 6x, put a 6 here. etc.
Volume (uL) Loaded per Well of Gel:
- This volume changes depending on well size and gel type, so fill in the volume you're planning to load per well.
#/Times Samples Will be Run:
- Enter how many times you will be running each sample. If you're running a sample 3 times, put a 3 here. This calculator has a built-in 10% surplus so you will have enough volume for all 3 runs + a bit extra.
Additional Dilution Factor
- When a “1” is in this cell the “Protein Loaded per Well (ug)” will be the highest it can be, given your sample concentrations. This means your lowest concentration sample will have 0 uL lysis buffer and be comprised entirely of reducing agent, Laemmle buffer, and your lysate sample. If the protein loaded per well (ug) is too high, tinker with this number until the amount loaded per well is what you'd like.

Step 4: Prep Your Samples

We're done inputting numbers! Proceed to the green shaded part of the chart and start prepping your samples! All samples will end up equal in volume and protein concentration.

Gel Loading Calculator for Western Blotting

Gel Loading Calculator for Western Blotting

Topics

DOI