TITLE: BCA analysis of hydrolyzable proteins in pollen using the Pierce BCA assay kit
AUTHORS: Mark J. Carroll

[DESCRIPTION]
This protocol quantifies total protein contents of small amounts of pollen (10 mg) using the bicinchoninic acid (BCA) assay of the Pierce BCA protein assay kit (Thermo Scientific product number 23225). This protocol adapts the kit protocol for analysis of anther pollen, corbicular pollen, and stored pollen from bee colonies. Pollen samples are subjected to acid hydrolysis to digest refractory proteins and to degrade sugars and other pollen substances that interfere with total protein assays. The acid hydrolysis releases substantially more protein from pollens than occurs with soluble proteins but may result in some protein losses due to the harsh acidic conditions. Similar BCA analysis methods without hydrolysis may be used to quantify soluble proteins (proteins obtained without acid hydrolysis or other digest methods) which may exclude insoluble pollen proteins from full quantification. The total protein contents obtained using BCA analysis of acid hydrosylates represents the maximum protein available to consumers and likely includes some refractory proteins that may not be digested by the consumer.

The assay is modified from the original standard protocol to work for pollen analysis:  Document Connect (thermofisher.com).  Please note that our steps differ from the commercial source protocol.


National Program Number: NP 305
Project Number: 2022-21000-022-000D, 2022-21000-022-053-I
Carl Hayden Bee Research Center, USDA-ARS PWA, Tucson, Arizona, USA
Grand Challenge - Assessing the Nutrient Contents of Pollen for Bees


[BEFORE_START]
Read the section on the handling of the liquid nitrogen-nitrogen atmosphere cooler. The method described here is both difficult and potentially hazardous given the need for pipetting near open liquid nitrogen. Check the resistance of the red rubber crimp caps to acid hydrolysis before attempting large scale processing of samples.

[GUIDELINES]
Sample contents are quantified by comparison against known amounts of BSA external standards.
The external standards are not processed through acid hydrolysis (as the samples are) but are subjected to the BCA reaction.
The working detection range of the BCA reaction is approximately 20 mg to 2000 mg per sample.
The 10 mg sample mass is based on dried protein estimates within this range.
Both sample and standard reaction solutions are run in triplicate on the 96 well plate.
The BCA reaction is time sensitive and continuous (no true end point) so maintain similar time intervals with each plate preparation.
External standards should be included on every BCA reaction plate.
If the sample is too concentrated, dilute spare acid hydrosylate and rerun the reaction.

[STEPS]
SECTION: Acid Hydrolysis of Pollen
1. Dry down the pollen sample in a freeze dryer.
SECTION: Acid Hydrolysis of Pollen
2. Weigh out 10.0 mg of pollen into a crimp cap vial. Place the sample vials into a 24 well plate (acts as a vial holder).
SECTION: Acid Hydrolysis of Pollen
3. Warning – read the precautions and hazards regarding handling of liquid nitrogen for creation of a nitrogen atmosphere.This step is potentially hazardous in several ways.

Set up a nitrogen atmosphere in a tall thin styrofoam cooler (at least 30 cm) by adding enough liquid nitrogen to cover the bottom about 1.5 cm deep. Cover the cooler with a lid when not in use. Place a small piece of styrofoam in the liquid nitrogen to act as a bench inside the cooler. The bench should be well grounded and not float or wiggle around in the liquid nitrogen. The goal is to create a cold nitrogen atmosphere around the vials yet avoid contact of the researcher or vials with liquid nitrogen.
SECTION: Acid Hydrolysis of Pollen
4. Place the open vials in their vial holder on the cooler bench and cover the nitrogen atmosphere cooler with the lid for 10 minutes. This sealing will allow air within the vial to be largely displaced by cold nitrogen gas.
SECTION: Acid Hydrolysis of Pollen
5. Carefully add 1000 µL concentrated (12M) hydrochloric acid to each vial in the cooler. Avoid spilling acid droplets on the vial lip where the crimp cap will be sealed. Cover the vials in the cooler again for 10 more minutes to allow the nitrogen atmosphere to enter the vials again.
SECTION: Acid Hydrolysis of Pollen
6. Carefully reach into the cooler and use a vial crimper to seal each vial securely with a red rubber crimp cap.
SECTION: Acid Hydrolysis of Pollen
7. Remove the vials from the cooler.Inspect the vials to ensure that the caps are well sealed. Double crimp the cap at two different angles to ensure a tight seal.
SECTION: Acid Hydrolysis of Pollen
8. Incubate the vials in a heating block on a hot plate at 110°C for 16 hours. Cover the heating blocks with aluminum foil to limit convention heat loss. Visually check the vial caps for leaks (corrosion will be notably visible) after the plate reaches temperature.
SECTION: Preparing Sample Protein Hydrosylates For the BCA Assay
9. Allow the vials and their heating blocks to cool to room temperature. Examine the vial caps for leakage and corrosion. Spin the vials down for a few seconds in a vacuum centrifuge (without vacuum) to draw down condensation.
SECTION: Preparing Sample Protein Hydrosylates For the BCA Assay
10. Pipette 300 µL of the 1000 µL acid digest into a new crimp vial. Re-seal and save the remainder of the vial hydrosylate as backup material.
SECTION: Preparing Sample Protein Hydrosylates For the BCA Assay
11. Completely remove all of the acid and water from the vials by evaporation in a vacuum centrifuge. Hydrochloric acid is volatile but requires several hours to evaporate.
SECTION: Preparing Sample Protein Hydrosylates For the BCA Assay
12. Reconstitute the dried material in 1000 µL PBS buffer. Vortex for 15 seconds, sonicate for 30 seconds, and vortex again for 15 seconds. Examine representative vial contents under a microscope to ensure that the material went into solution.
SECTION: Preparing Sample Protein Hydrosylates For the BCA Assay
13. Make a step dilution series of BSA (bovine serum albumin) standards from 0 to 2000 µg/mL using the BSA stock ampule (2000 µg/mL). Work up 500 µL standards for each concentration since the standards will be run in triplicate. Use the same exact batch of diluent solution (PBS buffer) as your samples. Add the following materials to a crimp cap vial, cap with a red rubber crimp cap, and briefly vortex.

target
conc. (µg/mL)    BSA solution (µL)    diluent solution (µL)
2000                    500 of 2000 µg/mL            0
1500                    373 of 2000 µg/mL            127
1000                    250 of 2000 µg/mL            250
750                      187.5 of 2000 µg/mL          312.5
500                      250 of 2000 µg/mL            750 (1000 µL total - use excess in later dilutions)
250                      250 of 500 µg/mL              250
125                       125 of 500 µg/mL              375
25                          25 of 500 µg/mL              475
0                              0                                      500
SECTION: BCA Total Protein Assay Reaction
14. Turn on the plate reader for sufficient time (30 minutes for a BioTek Synergy HT microplate reader) for the optics to reach operating temperature and stabilize. Set the plate reader temperature to 37°C if your plate reader has thermal control.

Each plate has sufficient wells to run 9 standard concentrations (27 wells total on the left side) and 23 samples (69 wells total) in triplicate.
SECTION: BCA Total Protein Assay Reaction
15. Prepare working reagent (WR in the kit manual) from kit Solutions A and B. For a full 96 well plate (200 µL per well, 19.2 mL total), add 392 µL Solution B to 20.000 mL Solution A to make 20.392 mL WR. This reagent lasts up to one week at room temperature.
SECTION: BCA Total Protein Assay Reaction
16. Add 200 µL working reagent (WR) to each well in a 96 well plate.
SECTION: BCA Total Protein Assay Reaction
17. Add 25 µL of standard or sample solution to each well to start the reaction.
SECTION: BCA Total Protein Assay Reaction
18. Incubate the plate(s) at 37°C for 30 minutes.

If multiple plates are run, record the time and maintain a uniform stagger for the incubation time and subsequent plate reading. The reaction is continuous and absorbance will increase over time.
SECTION: BCA Total Protein Assay Reaction
19. Place the plate into a plate reader and read the 562 nm absorbance for each well. Set the plate reader at 37°C if the reader has thermal control.
SECTION: Calculation of Total Protein from Absorbance
20. Export the 562 nm absorbance data. Calculate the average 562 nm absorbance for each sample and standard triplicate.
SECTION: Calculation of Total Protein from Absorbance
21. Generate a standard curve from the standards. Calculate the net 562 nm absorbance for each standard concentration as: 

562 nm absorbance of the standard concentration – 562 nm absorbance of the 0 µg/mL standard 
SECTION: Calculation of Total Protein from Absorbance
22. Plot the net 562 nm absorbance for each standard concentration against the standard protein concentration (ug protein/mL). Apply a linear best fit line with a zero intercept.
SECTION: Calculation of Total Protein from Absorbance
23. Calculate the average net 562 nm absorbance for each sample triplicate as:

562 nm absorbance of the sample concentration – 562 nm absorbance of the 0 µg/mL standard.

If the sample 562 nm net absorbance exceeds the 2000 µg/mL BSA protein standard net absorbance maximum, repeat the BCA reaction with a 1:4 diluted sample. Do not dilute the standards and use the same diluent solution (PBS solution) as the standards and other samples.
SECTION: Calculation of Total Protein from Absorbance
24. Calculate the amount of total protein present in each sample. Use the best fit line equation to calculate the amount of total protein present in each sample well. Steps that were performed differently between standards and samples (i.e. dilutions, taking only a fraction of the total sample) need corrections in calculations. Apply a correction factor to adjust for the reduced proportion of sample protein hydrosylate material used in the BCA analysis (300 µL of 1000 µL total, so 3.33x). Apply a dilution correction factor if samples were further diluted (i.e. a dilution of 1:4 (to 1/5x original concentration) requires a 5x dilution factor).
SECTION: Calculation of Total Protein from Absorbance
25. Divide the amount of total protein present by the pollen mass to obtain a total protein concentration for the pollen sample.