Calculations for Protein Gel Loading

This page is part of the SDS-PAGE lab, which includes these pages:

The whole experiment will be spread over three lab days. You will also use these methods as part of the pGLO lab.

When you run an SDS-PAGE gel, it's important to load an appropriate mass of protein in each lane. If there's too little protein, you might not see the bands you're looking for; if there's too much, the bands will smear together. In addition, you should load approximately the same mass of protein in each lane; otherwise, your gel may be distorted and difficult to interpret.

There's no general rule for how many micrograms of protein to load into one lane of a gel. The amount depends on various factors, including:

  • Characteristics of the gel and the staining technique you will use. We can keep these things constant in the Bio 6B lab.
  • The number of protein bands in the sample. If there are 25 bands, you will need to load enough protein to make each band visible. Suppose your staining technique is sensitive enough for you to see a band with 50 ng of protein (warning: this is only a guess). If you're loading a sample of purified protein containing only a single band, you might load 50 ng protein in each lane of the gel. If your sample contained 10 different bands, you'd need to load at least 500 ng total protein to give each band enough protein to be visible. In practice, when we run bacterial lysates on gels in the Bio 6B lab, it's best to load 4-8 μg of protein per lane.

Overview

You'll start with a sample of unknown protein concentration, so the preparation and calculations for protein gel loading will involve several steps:

  • Perform a protein assay and calculate the protein concentration of your sample.
  • Prepare your gel-ready protein sample and calculate its concentration.
  • Calculate the volume of gel-ready protein sample to load in each well.

It's a good idea to draw a flow diagram for the whole procedure to remind you where you are for each step.

Perform a protein assay and calculate the protein concentration of your sample

See SDS-PAGE Sample Preparation and Assay for detailed instructions on how to perform the protein assay. This page only covers the calculations.

The protein assay measures the protein concentration in your assay tube, after it has been mixed with working solution. Once you know the protein concentration in your assay tube, you can calculate the concentration in your original sample tube as follows:

C1V1=C2V2

Where:

  • C1: Sample tube concentration. This is what you'll calculate.
  • V1: Volume of sample added to the assay tube (you'll normally start with 10 μl, but you might need to change it if your readings are too high or too low).
  • C2: Assay tube concentration. You'll read this from the Qubit fluorometer. Be sure to write down the units when you record this number.
  • V2: Assay tube volume. For Qubit assays, this will always be 200 μl; that's how the fluorometer is designed to work.

In fact, you can get the Qubit to perform this calculation for you, but you should know how to do it yourself. Once you've done the calculation, take a moment to see if your answer makes sense. In a standard assay with 10 μl plus 190 μl working solution, your sample tube concentration should be 20 times your assay tube concentration.

Prepare your gel-ready sample and calculate its concentration

Your protein sample is typically a lysate -- the water-soluble contents that are released when cells are burst open (lysed). The lysate contains a wide range of macromolecules. You'll load all of these molecules onto your protein gel, but in the end you'll see only the proteins because you'll use a protein-specific stain.

As described in on the electrophoresis page, you need to denature your proteins and give them a consistent negative charge density for SDS-PAGE. To make your protein sample ready for SDS-PAGE, you need to mix it as follows:

65 μl  Protein sample
25 μl  4x LDS sample buffer
10 μl  Sample reducing agent
100 μl   Total

Be sure that the ingredients are fully mixed (the purple color should be evenly distributed) and be careful not to put bubbles into it (the SDS detergent tends to get frothy). After you prepare the gel-ready samples, you need to heat them at 70° for 10 minutes before loading on the gel.

These amounts will be the same for all the protein samples you do this quarter. The final concentration of sample buffer will be 1x. See the lab manual for more detail on what these ingredients do. Be sure to follow the instructions in the lab manual for heating the sample before loading it on the gel. This page is only about the calculations.

Calculate the protein concentration of your gel-ready sample

Use the familar equation:

C1V1=C2V2

This time,

  • C1: Sample tube concentration. This was determined in the previous calculation, based on your assay.
  • V1: Volume of protein sample added to the gel-ready tube (65 μl).
  • C2: Gel-ready tube concentration. This is what you're calculating.
  • V2: Gel-ready tube volume: 100 μl.

Calculate the volume of gel-ready sample to load in each well

Now that you know the concentration of the sample you'll be loading on the gel, you can calculate the volume to pipet into the well. The recommended mass of protein may vary for different experiments, but for your first gel with fish muscle proteins, try to get 4 μg protein in each lane. Your calculation will look like this (if the gel-ready protein sample concentration is b μg/μl):

Note that you need to invert the concentration to make the units cancel out. You need an answer in microliters, because that's how our micropipets work. If your protein concentration is in μg/ml, you can put a conversion factor into your equation like this:

In the end, you hope that your answer is less than 25 μl, because that's all that will fit in one well of the gel.

What if you can't fit 4 μg of protein into the well?

The wells on our SDS-PAGE gels hold 25 μl. If you calculate that you'd need more than 25 μl to provide 4 μg of protein, you'll have to load as much as you can and hope for the best. You can calculate the maximum mass of protein that you'll be able to fit in the gel like this:

If the total amount is below a few hundred nanograms, you might not see much on your gel. Don't get depressed yet, though — maybe your assay is wrong and you have more protein than you think. This is why it's important to compare your results to those of other groups in the lab. Finally, even if the assay tells you that there is a low protein concentration, go ahead and complete the experiment. The truth is in the gel.

Review

Concepts

  1. How would you know how much protein to load on a gel?

Calculations

  1. Suppose you’re running a protein gel. You perform a protein assay on your sample, with 20 µl protein sample and 180 µl working solution. You measure a protein concentration of 40 µg/ml in the assay tube. What is the protein concentration in your sample tube (in µg/ml)?
  2. Continuing the same sample, suppose you mix 65 µl of protein sample, 25 µl of sample buffer, and 10 µl of sample reducing agent in a new tube (gel-ready). What is the protein concentration in your gel-ready tube (in µg/ml)?
  3. Continuing the same gel-ready sample, suppose you load 20 µl of protein on the gel. How much protein is that (in µg)?
  4. Continuing with the same gel-ready sample, suppose you want to load 6.0 µg of protein on the gel. How much is that in µl?

 

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