Protein Analysis in Large Benthic Foraminifera

Abstract

Large benthic foraminifera (LBFs) have long been used as environmental recorders of ocean chemistry. Although the importance of foraminifera in paleo-reconstructions of ancient oceans and as sediment producers is well documented, the biology of tropical symbiont-bearing foraminifera has only recently gained increased attention. Tropical symbiont-bearing LBFs represent a unique and important subset of LBFs in that they are vital to coral-reef ecosystems and host a wide suite of algal symbionts (e.g., dinoflagellates, diatoms, red algae, green algae and cyanobacteria). Previous studies on both host and symbiont physiology have been performed in order to gauge the foraminiferal response to a variety of stressors, including elevated temperature and nutrient levels, as well as acidification. Recently, protocols have been developed for protein analysis in LBFs that will allow for expression analyses of target proteins from both members of the holobiont. In this chapter, we detail a protein expression protocol for one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1-D SDS-PAGE) and consequent western blotting for determination of protein expression in the foraminiferal holobiont. This technique has the potential to target proteins that are specific to either host or symbiont compartments, a breakthrough that may ultimately allow for an increased understanding of the molecular-scale regulation of the symbiosis that is vital to these globally important calcifiers.

Appendix 1

	Chemical formula	Final concentration	Amount	Manufacturer
RIPA buffer a
1 M Tris–HCl (pH 6.8)	NH2C(CH2OH)3	50 mM	2.5 mL	JT Baker 4099-02
NP-40/Triton X 1 %	C14H22O(C2H4O)n(n=9-10)	1 %	500 μL	Sigma-Aldrich X-100
Sodium-deoxycholate	NaC24H39O4	0.25 %	0.25 g	Thermo Scientific 89904
5M Sodium chloride	NaCl	150 mM	1.5 mL	Sigma-Aldrich 31434
Double-distilled water (ddH2O)	H2O		To 50 mL
Protease inhibitor	Inhibitor Cocktail	per manufacturer's instruction		Roche Complete Protease Inhibitor Cocktail Tablets 04693116001
		Total	50 mL
2× SDS-PAGE Laemmli sample buffer b
1M Tris–HCl (pH 6.8)	NH2C(CH2OH)3	125 mM	1.25 mL	JT Baker 4099-02
10 % SDS	NaCH3(CH2)11OSO3	4 % (w/v)	4 mL	JT Baker 4095-02
80 % Glycerol	HOCH2CH(OH)CH2OH	20 % (w/v)	2.5 mL	Sigma-Aldrich 15523
2 mg/mL Bromophenol blue	C19H9Br4NaO5S	0.01 % (w/v)	100 μL	Sigma-Aldrich RD32768
2-Mercaptoethanol	HSCH2CH2OH	5 %	500 μL	Sigma-Aldrich M7154
ddH2O	H2O	–	To 10 mL	–
		Total	10 mL
10 % SDS c
10 % Sodium dodecyl sulfate (SDS)	NaCH3(CH2)11OSO3	10 % (w/v)	25 g	JT Baker 4095-02
ddH2O	H2O		To 250 mL
		Total	250 mL
10× SDS-PAGE running buffer d
Tris-base	NH2C(CH2OH)3	0.25 M	30.3 g	JT Baker 4099-02
Glycine	NH2CH2COOH	1.92 M	144.0 g	Sigma-Aldrich G8898
SDS	NaCH3(CH2)11OSO3	33 mM	10.0 g	JT Baker 4095-02
ddH2O	H2O	–	To 1,000 mL
		Total	1,000 mL
1 M Tris–HCl (pH 6.8) e
Tris-base	NH2C(CH2OH)3	1 M	60.57 g	JT Baker 4099-02
12 N HCl	HCl	–	varies
ddH2O	H2O	–	To 500 mL
		Total	500 mL
1.5 M Tris–HCl (pH 8.8) f
Tris-base	NH2C(CH2OH)3	1.5 M	90.86 g	JT Baker 4099-02
12N NaOH	NaOH	–	varies
ddH2O	H2O	–	To 500 mL
		Total	500 mL
10 % APS g
Ammonium persulfate (APS)	(NH4)2S2O8	10 % (w/v)	0.5 g	JT Baker 0762-01
ddH2O	H2O	–	To 5 mL
		Total	5 mL
Bromophenol blue h
Bromophenol blue	C19H9Br4NaO5S	2 mg/mL	10 mg	Sigma-Aldrich RD32768
ddH2O	H2O	–	To 5 mL
		Total	5 mL
Gel Fixation Buffer prior to SYPRO ® Ruby Stain i
Methanol	CH3OH	50 % (v/v)	500 mL	Sigma-Aldrich 34966
Acetic acid	CH3COOH	7 % (v/v)	70 mL	Sigma-Aldrich A6283
ddH2O	H2O	–	To 1,000 mL
		Total	1,000 mL
Destaining buffer for SYPRO ® Ruby Stain
Methanol	CH3OH	10 % (v/v)	100 mL	Sigma-Aldrich 34966
Acetic acid	CH3COOH	7 % (v/v)	70 mL	Sigma-Aldrich A6283
ddH2O	H2O	–	To 1,000 mL
		Total	1,000 mL
5× SDS-PAGE transfer buffer j
Tris-base	NH2C(CH2OH)3	0.123 M	15.0 g	JT Baker 4099-02
Glycine	NH2CH2COOH	0.959 M	72.0 g	Sigma-Aldrich G8898
ddH2O	H2O	–	Fill to 1,000 mL
		Total	1,000 mL
1× SDS-PAGE transfer buffer k
Methanol	CH3OH	20 % (v/v)	200 mL	Sigma-Aldrich 34966
5× transfer buffer	–	20 % (v/v)	200 mL	–
ddH2O	H2O	–	To 1,000 mL
		Total	1,000 mL
10× TBS buffer l
Tris-base	NH2C(CH2OH)3	0.1M	12.11 g	JT Baker 4099-02
Sodium chloride	NaCl	1.5M	87.66 g	Sigma-Aldrich 31434
12N HCl	–	–	varies
ddH2O	H2O	–	To 1,000 mL
		Total	1,000 mL
1× TBST Buffer m
10× TBS	–	10 % (v/v)	100 mL	–
Tween-20	C58H114O26	0.5 % (v/v)	500 μL	Sigma-Aldrich P5927
ddH2O	H2O	–	To 1,000 mL	–
		Total	1,000 mL
5 % blocking buffer
1× TBST buffer	–	–	10 mL
Skim milk	–	5 % (w/v)	0.5 g	Sigma-Aldrich Fluka 70166
		Total	10 mL

1. ^aInstructions: Add the protease inhibitor right before use, as protease inhibitors will degrade upon continual storage in liquid form. Store RIPA lysis buffer at 4 °C
2. ^bInstructions: Add all components and keep at −20 °C for use within 3 weeks. Add 2-Mercaptoethanol in a fumehood, and avoid repeated freeze-thaws
3. ^cInstructions: Dissolve 25 g SDS in 200 mL ddH2O, then fill to 250 mL
4. ^dInstructions: Dissolve Tris-base, glycine and SDS in 900 mL ddH₂O, then fill to 1,000 mL. Dilute to 1× with ddH₂O before use in gel electrophoresis
5. ^eInstructions: Dissolve Tris-base in 400 mL ddH₂O, adjust pH to 6.8 with 12 N HCl and fill to 500 mL with ddH₂O
6. ^fInstructions: Dissolve Tris-base in 400 mL ddH₂O, adjust pH to 8.8 with 12N NaOH, and fill to 500 mL with ddH₂O
7. ^gInstructions: Dissolve APS powder in 5 mL ddH₂O. Aliquot into 1.5 mL eppendorff tubes, and store at −20 °C for a maximum of 4 weeks. Avoid repeated freeze-thaws
8. ^hInstructions: Dissolve bromophenol blue in 5 mL ddH₂O. Aliquot into 1.5 mL eppendorff tubes, and store at −20 °C
9. ⁱInstructions: Add and mix chemicals in the fume hood. Rapid Protocol: (1) Microwave gel for 30 s, agitate (70 rpm) for 30 s. (2) Microwave gel for 30 s, agitate (70 rpm) for 5 min. (3) Microwave gel for 30 s, agitate (70 rpm) for 25 min. (4) Continue with Step 4 in Sect. 5.3.5
10. ^jInstructions: Dissolve Tris-base and glycine in 900 mL ddH₂O, then fill to 1,000 mL
11. ^kInstructions: Make up beforehand, and store at 4 °C
12. ^lInstructions: Dissolve tris-base and sodium chloride in 900 mL ddH₂O, adjust pH with 12 N HCl to pH 7.6, and fill to 1,000 mL
13. ^mInstructions: Mix TBS, ddH₂O and Tween-20, and mix well